Inhibitors of glycosaminoglycans

ABSTRACT

The present invention provides peptide derivatives with a specific affinity for glycosaminoglycan molecules. These peptide derivatives include multimers as well as chemically modified peptides and may be prepared by a variety of methods. The peptides of the invention have numerous functions, including but not limited to use as inhibitors of glycosaminoglycan-mediated signaling events and targeting agents. Peptides of the invention may be directed against any glycosaminoglycan, including hyaluronic acid, chondroitin sulfate A, chondroitin sulfate C, dermatan sulfate, heparin, keratan sulfate, keratosulfate, chitin, chitosan 1, and chitosan 2. The peptide derivatives of the invention also have therapeutic uses in the treatment and prevention of diseases involving inflammatory diseases, cancer, and cancer metastasis, autoimmune diseases, etc.

CROSS-REFERENCE TO RELATED APPLICATIONS

[0001] This application is a continuation-in-part application of U.S.patent application Ser. No. 09/532,709 entitled MODULATORS OFPOLYSACCHARIDES AND USES THEREOF, filed Mar. 22, 2000 and claimspriority to U.S. provisional Patent Application No. 60/277,790 entitled“INHIBITORS OF GLYCOSAMINOGLYCANS” filed Mar. 21, 2001 and thedisclosures of these applications are hereby incorporated by referencein their entirety into this application for all purposes.

GOVERNMENT RIGHTS

[0002] The government may have certain rights in the present inventionpursuant to grant number R03 AR47402 from the National Institutes ofHealth.

FIELD OF THE INVENTION

[0003] The present invention relates generally to the fields of cancer,immunology and inflammatory diseases. More particularly, it concernspeptide inhibitors of glycosaminoglycans. The invention also providestherapeutic and preventive methods for the treatment of inflammatorydiseases, autoimmune diseases and other glycosaminoglycan-associateddiseases. Additionally, the invention provides anticancer therapiesusing glycosaminoglycan binding agents.

BACKGROUND OF THE INVENTION

[0004] Interactions of cells of the immune system with components of theextracellular matrix (ECM) are responsible for the induction of variousimmune responses including inflammatory responses. In addition to beingan important component of the extracellular structure, the ECM also isinvolved in cellular signal transduction events by interactions withcellular receptors. Thus, the ECM modulates cell adhesion, cellproliferation, cell differentiation, etc. (Schubert et al. Trends Cell.Biol., 2:63-66, 1992). Major constituents of the ECM includeglycosaminoglycans, fibronectin, laminin, collagens, and proteoglycans,which bind specific cell surface receptors via protein-protein andprotein-carbohydrate interactions. The glycosaminoglycans are linearpolymers of repeating disaccharides often bound covalently to a proteincore.

[0005] Hyaluronan (also known as hyaluronic acid or hyaluronate) (HA),is a glycosaminoglycan lacking a protein core, and is one of the majornon-structural elements of the extracellular matrix (Laurent et al.,F.A.S.E.B. J. 6: 2397-2404, 1992; Aruffo et al. Cell. 61:1303-1313,1990; Culty et al., J. Cell. Biol. 111:2765 1990; Underhill et al. Cell.Sci. 103, 293-298, 1992; Toole et al. Plenum Press, New York,1384-1386,1991). HA also is expressed on cell surfaces and has beenshown to bind several different molecules, including CD44 (Aruffo et al.Cell. 61:1303-1313, 1990; Miyake et al. J.Exp.Med. 172:69-75, 1990), thereceptor for HA-mediated motility (RHAMM) (Hardwick et al. J.Cell.BioL117:1343-1350, 1992), link protein (Hardingham et al. Biochem.J.177:237-247, 1979), aggrecan (Watanabe et al. J.Biol.Chem.272:28057-28065, 1997), versican (LeBaron et al. J.Biol.Chem.267:10003-10010, 1992), hyaluronectin (Delpech et al. J.Neurochem.36:855-859, 1981), neurocan (Rauch et al. J.Biol.Chem., 267:19536-19547,1992), liver sinusoidal endothelial HA receptor, inter-α-trypsininhibitor-related proteins (10), BEHAB (brain-enriched HA binding),CD38, lymphatic vessel endothelial HA receptor 1, and white fat/bonemarrow/osteoblast HA binding proteins. Conversely, CD44 binds not onlyHA, but also collagens, fibronectin, chondroitin sulfates, heparin,heparin sulfate, and serglycins. Thus, although CD44 (or HA) isgenerally considered to be a primary HA receptor (or a principal CD44ligand), HA-CD44 interaction represents one of the multiple mechanismsby which HA and CD44 may regulate cellular activities.

[0006] HA is a repeating disaccharide of [GlcNAcβ1-4GlcUAβ1-3]_(n) thatexists in vivo as a high molecular weight linear polysaccharide. HA isfound in mammals predominantly in connective tissues, skin, cartilage,and in synovial fluid, and is also the main constituent of the vitreousof the eye. In connective tissue, the water of hydration associated withHA creates spaces between tissues, thus creating an environmentconducive to cell movement and proliferation. HA plays a key role inbiological phenomena associated with cell motility including rapiddevelopment, regeneration, repair, embryogenesis, embryologicaldevelopment, wound healing, angiogenesis, and tumorigenesis (Toole etal. Plenum Press, New York, 1384-1386,1991; Bertrand et al. Int. J.Cancer. 52:1-6, 1992; Knudson et al. F.A.S.E.B. J. 7:1233-1241, 1993).HA levels have been shown to correlate with tumor aggressiveness (Ozelloet al. Cancer. Res. 20:600-604, 1960; Takeuchi et al. Cancer. Res.36:2133-2139, 1976; Kimata et al. Cancer. Res. 43:1347-1354, 1983), andcan be indicative of the invasive properties of tumor cells (Knupfer etal. Anticancer. Res. 18:353-6, 1998).

[0007] HA also is involved in immune responses, for example, increasedbinding of HA to one of its receptors, CD44, has been shown to mediatethe primary adhesion (“rolling”) of lymphocytes to vascular endothelialcells under conditions of physiologic shear stress, and this interactionmediates activated T cell extravasation into an inflamed site in vivo inmice (DeGrendele et al. J.Exp.Med. 183:1119-1130, 1996; DeGrendele etal., J. Immunol. 159:2549-2553, 1997; DeGrendele, et al., Science.278:672-675, 1997b). Alterations in levels of HA and otherglycosaminoglycans have also been associated with unwanted immuneresponses, and in diseases and disorders such as rheumatoid arthritis,atopic dermatitis, psoriasis, multiple sclerosis, transplantationrejection. For example, HA and other glycosaminoglycans display arealtered in autoimmune disorders such as arthritis, and decreased levelsof both hyaluronic acid and chondroitin 6-sulfate have been found in thediseased synovial fluid of both adults with rheumatoid arthritis(Bensouyad et al. Ann. Rheum. Dis. 49:301-307, 1990) and children withjuvenile rheumatoid arthritis (Spelling et al. Clin. Exp.Rheumatol.9:195-9, 1991).

[0008] Dendritic cells (DC) play essential roles in the induction ofcellular immune responses to a variety of relevant antigens. DC areknown to play critical roles in the induction of cellular immuneresponses against a wide variety of antigens of relevance, includingchemical haptens, foreign proteins, infectious microbes, andtumor-associated antigens (Steinman et al. Ann. Rev. Immunol. 9:271,1991; Stingl et al. ed. McGraw Hill and Co. New York, p. 172, 1993).Interaction between HA, expressed on endothelial cells, and CD44,expressed on activated dendritic cells as well as T cells, andgranulocytes, is believed to mediate homing of such leukocytes to theirtarget sites.

[0009] Glycosaminoglycans, particularly HA, also are known to mediateother cellular interactions that involve binding and entry into a cell.For example, HA is involved in infection of mammalian cells by the HumanImmunodeficiency Virus (HIV), since HIV is known to bind to HA uponinfection. Both HA and monoclonal antibodies to its receptor CD44 werefound to inhibit HIV infection of monocytes by monocytotropic HIV(Levesque et al. J. Immunol. 156:1557-65, 1996). HA also is involved inmammalian zygote formation by mediating binding of the oocyte and thesperm. Data indicate that HA in the cumulus matrix may act to prime thefertilizing sperm for induction of the acrosome reaction by constituentsof the cumulus and/or zona pellucida. HA is thought to mediate thisinteraction by binding to the PH-20 protein to increase basal levels ofintracellular calcium and thereby potentiate the acrosome reaction(Sabeur et al. Zygote. 6:103-11, 1998). HA mediates sperm motility byenhancing phosphorylation of proteins including HA binding protein(Ranganathan et al. Cell. Mol. Biol. Res. 41:467-76, 1995).

[0010] Other important glycosaminoglycans involved in a variety ofphysiological and pathological states include, chondroitin sulfate A,chondroitin sulfate C, dermatan sulfate, heparin, keratan sulfate,keratosulfate, chitin, chitosan 1, and chitosan 2. These and otherglycosaminoglycans, and particularly hyaluronic acid, play importantroles in such varying physiological processes make them attractivetargets for therapeutic agents. However, glycosaminoglyeans have beenfound to be nearly non-antigenic, and hence, very few antibodies thatrecognize glycosaminoglycans have been isolated. Due to the lack ofantigenicity, it has been technically difficult to develop inhibitors orprobes of glycosaminoglycans. As such, there is a need in the art forinhibitors of glycosaminoglycan-mediated processes, and in particularfor inhibitors of hyaluronic acid-mediated processes.

SUMMARY OF THE INVENTION

[0011] Hyaluronic acid and salts play key roles in biological phenomenaassociated with cell motility including development, regeneration,repair, embryogenesis, embryological development, wound healing,angiogenesis, and tumorigenesis and immune responses. The presentinvention provides methods and compositions comprising artificialpeptide multimers that have the ability to bind HA with a bindingaffinity K_(a) of 5×10⁵ l/mol or more and inhibit HA-dependentprocesses. In particular, several different aspects of the immunesystem, including inhibiting inflammatory reactions, inhibition ofcytokine release, inhibition of antigen presentation, inhibition ofclonal expansion of T-cells, inhibition of maturation ofantigen-presenting cells (APCs) such as dendritic cells, inhibition ofAPC-T cell cluster formation, inhibition of T-cells activation andinhibition of cell adhesion, leucocyte extravasation, etc can bemanipulated by the invention. The invention further provides therapeuticand preventive methods for the treatment of inflammatory diseases,autoimmune diseases and other glycosaminoglycan-associated diseases.Additionally, the invention provides anticancer therapies that inhibittumorigensis and metastasis.

[0012] In a first embodiment, an artificial peptide multimer isdisclosed which comprises the structure (Z)_(n)X(Y)_(m) where X is anyamino acid and each of Y and Z is an aliphatic or polar aliphatic aminoacid of between 6 and 30 amino acid residues. The peptide multimer willbind a glycosaminoglycan or fragment thereof with a binding affinityK_(a) of 5×10⁵ l/mol or more. The structure (Z)_(n)X(Y)_(m) is comprisedwithin a subunit of the multimer. The multimer may have two or morepeptide units with the same amino acid sequence, or with different aminoacid sequences. The multimer may be a dimer, a trimer, a tetramer, apentamer, a hexamer, a heptamer, an octamer, a nonamer, decamer orhigher order multimer. The glycosaminoglycan bound may be hyaluronicacid or a salt thereof, chondroitin sulfate, chondroitin sulfate C,dermatan sulfate, heparin, keratan sulfate, keratosulfate, chitin,chitosan 1 or chitosan 2.

[0013] The multimer subunits may be connected by one or more linkermolecules, such as amino acids (peptide linkers) or non-peptide linker(e.g., succinic acid, polyethylene glycol (PEG)). The peptide subunitsmay comprise a motif ZZZXZZZ, wherein Z is an amino acid selected fromthe group consisting of aliphatic and polar aliphatic residues, andwherein X is any amino acid. The subunit may further comprises an N- orC-terminal extension W_(p), wherein W is any basic or neutral aminoacid, and p is an integer between 3 and 13, for example, wherein W isarginine or glycine independently. The peptide subunit also may comprisea terminal serine. In a particular embodiment, the extension is aC-terminal GGGS.

[0014] In another embodiment, the peptide multimer may be chemicallymodified. The chemical modification comprises amidation, PEGylation,glycosylation, acetylation, prenylation, phosphorylation, biotinylation,carboxylation, carbonylation, or may be further derivatized by additionof known protecting/blocking groups (substitution with “bulky” sidechains, e.g., methyl for alpha-hydroxyl). Other modifications includeuse of D-amino acids, cyclization, use of trans-olefin bonds. Inparticular embodiments, one or more of the peptide multimer subunitscomprises the peptide sequence GAXWQFXALTVX (SEQ ID NO: 1), wherein X isany amino acid. In a specific embodiment, the multimer comprises allGAHWQFNALTVR (SEQ ID NO: 2) subunits. In a particular embodiment, theextension is a C-terminal GGGS.

[0015] In another embodiment, there is provided a pharmaceuticalcomposition comprising (a) an artificial peptide multimer comprising thestructure (Z)_(n)X(Y)_(m) where X is any amino acid and each of Y and Zis an aliphatic or polar aliphatic amino acid of between 6 and 30 aminoacid residues and having the ability to bind a glycosaminoglycan orfragment thereof with a binding affinity K_(a) of 5×10⁵ l/mol or more;and (b) a pharmaceutically acceptable carrier, diluent or excipient.

[0016] In a further embodiment, there is provided a method of inhibitinga glycosaminoglycan-mediated reaction comprising administering to asubject an agent that binds a glycosaminoglycan or fragment thereof witha binding affinity K_(a) of 5×10⁵ l/mol or more, preferably a K_(a) of1×10⁶ l/mol or more, more preferably a K_(a) of 1×10⁷ l/mol or more, oreven more preferably a K_(a) of 1×10⁸ l/mol or more, or 1×10⁹ l/mol ormore, or 1×10¹⁰ l/mol or more, or 1×10¹¹ l/mol or more. The reaction maybe an inflammatory reaction, such as the interaction of an antigenpresenting cell such as a dendritic cell or macrophage and a T cell. Theactivity may comprise alteration in the secretion of immunoregulatoryfactors, cellular locomotion, cell-to-cell interaction, cell adhesion,cellular differentiation and maturation, cell growth and death, orinflammatory/immune reaction. Administration may be topical or systemicadministration, or local or regional to an affected body part or organ.Additionally, the peptide may be chemically modified as discussed above.

[0017] In still yet a further embodiment, there is provided a method fortreating or preventing cancer, tumorigenesis or cancer metastasiscomprising administering to a subject in need thereof an agent havingthe ability to bind a glycosaminoglycan or fragment thereof with abinding affinity K_(a) of 5×10⁵ l/mol or more. The agent may be anartificial peptide multimer comprising the structure (Z)_(n)X(Y)_(m)where X is any amino acid and each of Y and Z is an aliphatic or polaraliphatic amino acid of between 6 and 30 amino acid residues and havingthe ability to bind a glycosaminoglycan or fragment thereof with abinding affinity K_(a) of 5×10⁵ l/mol or more. The cancer may be braincancer, lung cancer, throat cancer, esophageal cancer, cancer of thehead and neck, skin cancer, breast cancer, stomach cancer, colon cancer,cancer of the rectum, cervical cancer, prostate cancer, ovarian cancer,liver cancer, pancreatic cancer or a cancer of the blood. The treatmentmay comprise inhibiting proximal or distal metastasis in a patientalready suffering from cancer. The peptide may be administered local orregional to a solid tumor, or systemically. The peptide may beadministered in conjunction with a second anti-cancer therapy, such asradiation, chemotherapy, or gene therapy. The second anti-cancer therapymay be administered concurrent, prior to or after the peptide. Theglycosaminoglycan may be hyaluronic acid, chondroitin sulfate,chondroitin sulfate C, dermatan sulfate, heparin, keratan sulfate,keratosulfate, chitin, chitosan 1 or chitosan 2. The peptide may be amonomer or a multimer comprising two or more peptide subunits asdiscussed above. The peptide also may be chemically modified, also asdiscussed above. The peptide may comprise a motif ZZZXZZZ, wherein Z isan amino acid selected from the group consisting of aliphatic and polaraliphatic residues, and wherein X is any amino acid. In a specificembodiment, the peptide sequence may be GAXWQFXALTVX (SEQ ID NO: 1),wherein X is any amino acid. In a particular embodiment, the extensionis a C-terminal GGGS.

[0018] In another embodiment, there is provided a method of treating orpreventing cancer comprising administering to a subject in need thereofa compound that modulates the synthesis, secretion or degradation of aglycosaminoglycan. The glycosaminoglycan may be hyaluronic acid,chondroitin sulfate, chondroitin sulfate C, dermatan sulfate, heparin,keratan sulfate, keratosulfate, chitin, chitosan 1 or chitosan 2. Thetreatment may comprise inhibiting metastasis in a patient alreadysuffering from cancer. The peptide may be administered local or regionalto a solid tumor, or administered systemically. The method also maycomprise a second anti-cancer therapy.

[0019] In still another embodiment, there is provided a method ofscreening for an anti-cancer agent comprising (a) providing a cell thatexpresses a glycosaminoglycan; (b) contacting the cell with a candidatesubstance; and (c) measuring the synthesis, secretion, degradation,surface expression or function of the glycosaminoglycan, wherein achange in the synthesis, secretion, degradation, surface expression orfunction of a glycosaminoglycan, as compared to a similar cell nottreated with the candidate substance, identifies the candidate substanceas an anti-cancer agent.

[0020] In still yet another embodiment, there is provided a method oftargeting an agent to a glycosaminoglycan structure comprising (a)providing a conjugate comprising a targeting moiety linked to the agent;and (b) contacting the conjugate with the cell. The targeting moiety maybe covalently linked to the agent. The cell may be located in a subject,for example, a human subject. The glycosaminoglycan may be expressed ona cell or contained within extracellular matrix. The agent may be atherapeutic agent, such as a radionuclide or cancer chemotherapeutic, ora diagnostic agent. Also, it may be a hyaluronidase. The cell may be acancer cell, and the glycosaminoglycan may be chondroitin sulfate,chondroitin sulfate C, dermatan sulfate, heparin, keratan sulfate,keratosulfate, chitin, chitosan 1 or chitosan 2.

BRIEF DESCRIPTION OF THE DRAWINGS

[0021] The following drawings form part of the present specification andare included to further demonstrate certain aspects of the presentinvention. The invention may be better understood by reference to one ormore of these drawings in combination with the detailed description ofspecific embodiments presented herein:

[0022] FIGS. 1A-B. (FIG. 1A) ³⁵S-labeled BW5147 thymoma cells wereincubated on HA-coated plates (0.1 μg/ml) that had been pretreated withthe indicated peptides (500 μg/ml). Data shown are the means+/−S.D. of %binding from quadruplicate samples. (FIG. 1B) FITC-conjugated HA (0.3μg/ml) was preincubation with the indicated peptide (500 μg/ml) andexamined for binding to BW5147 thymoma cells. Data shown are the bindingof FITC-conjugated HA in the presence of the indicated peptides (closedhistograms) as compared to the background autofluorescence levels (openhistograms).

[0023] FIGS. 2A-B. Improved biological activities of Pep-1 dimer andtetramer. Three different Pep-1 derivatives were compared for theirbiological activities at the same concentration in terms of the molarity(150 μM) of the 16-mer Pep-1 peptide sequence. HA-coated plates (0.1μg/ml) were pre-treated for 3 hr with Pep-1 (249 μg/ml), Pep-1 dimer(258 μg/ml) (FIG. 2A), or Pep-1 tetramer (998 μg/ml) (FIG. 2B).³⁵S-labeled B16-F10 melanoma cells were then examined for their bindingto these plates. Data shown are the means+/−S.D. of % binding fromquadruplicate samples. Asterisks indicate statistically significantdifferences (**p<0.01 by two-tailed Student's t-test).

[0024]FIG. 3. Impact of multimeric Pep-1 derivatives on hapten-inducedLC migration. BALB/c mice received two local injections of the indicatedPep-1 derivatives in the right ear (closed circles) and the indicated RPderivatives in the left ear 24 and 3 hours before topical application of0.5% DNFB on both ears. All the peptides were administered at theidentical dose in terms of the molarity (24 mmol/injection) of the16-mer Pep-1 peptide sequence, i.e., 40 μg/injection/ear for monomericPep-1 and RP, 41 μg/injection/ear for dimeric Pep-1 and RP, or 160μg/injection/ear for PEG-conjugated tetrameric Pep-1 and RP. Ear skinsamples were harvested at the indicated time points after DNFB paintingand examined for surface densities of IA⁺ LC. Data shown are themeans+/−s.e.m.(n=3) of % LC emigration as calculated by dividing fromthe formula: [(LC number before DNFB application−LC number after DNFBapplication)/LC number before DNFB application]×100. Asterisks indicatestatistically significant differences between the Pep-1 and the RPpanels (**p<0.01 by two-tailed Student's t-test).

[0025] FIGS. 4A-C. Impact of Pep-1 on lung metastasis of melanoma. (FIG.4A) B16-F10 melanoma cells were suspended (4×10⁵ cells/ml) in PBScontaining Pep-1 monomer (2 or 3 mg/ml) (circles) or 2% DMSO alone(triangles). C57BL/6 mice received i.v. injection of these cellsuspensions (500 μl/animal) and were examined for survival. Data shownare the cumulative survival curves from four independent experiments(n=37 for Pep-1 and n=20 for PBS control). Statistically significantdifference (p<0.01 by the log-rank test) was observed between the twopanels. (FIG. 4B) B16-F10 melanoma cells (1×10⁵ cells/animal) were s.c.injected into the back of C57BL/6 mice. Pep-1 or RP (40 μg/injection)was injected locally into the tumor inoculation sites 0. 3. and 6 daysafter tumor injection. Data shown are the means+/−s.e.m. (n=10) of tumorsizes and tumor weights measured 10 days after tumor inoculation. (FIG.4C) B16-F10 melanoma cells (2×10⁵ cells/well) were cultured in thepresence of the indicated concentrations of Pep-1 (closed circles) or PR(open circles). Data shown are the means+/−s.e.m.(n=3) of ³H-thymidineuptake at 24 hr. FIGS. 5A-C. Impact of Pep-1 on HA degradation products.(FIG. 5A) 96 well plates were coated with biotinylated-HA (0.1 μg/ml)and pretreated with 500 μg/ml of Pep-1 (open circles), RP (closedcircles), or PBS alone (triangles) for 3 hours. These plates were thenincubated for 30 min in the presence of HAase (from bovine testis).After extensive wash, the amounts of remaining biotinylated HA on theplates were determined by addition of streptavidin-alkaline phosphatasefollowed by p-nitrophenyl phosphate substrate. The % HA digestion wascalculated from the optical densities. The data shown are themeans+/−s.e.m.from triplicate samples analyzed with a hyperbolic model.(FIG. 5B) The impact of Pep-1 on HA digestion and on cell adhesion wasexamined in parallel using the HA-coated plates prepared simultaneously.Pep-1, RP, or PBS alone was added to the HA-coated plates as above andtheir effects on HAase-mediated HA degradation and on the adhesion of³⁵S-labeled BW5147 thymoma cells were compared. The data shown are themeans+/−s.e.m.(n=3) of 50% inhibition doses of HAase (left) and % celladhesion (right). Pep-1 showed significant (p<0.01 by ANOVA) inhibitionof cell adhesion, but not of HA digestion. (FIG. 5C) Low molecularweight HA fragments were prepared by sonication of HMW HA followed byovernight digestion with HAase at 37° C. The resulting HA fragments (50μg/ml) were pre-treated for 3 hours at 37° C. in RPMI medium containing0.5% DMSO with or without Pep-1 or RP (500 μg/ml) and then added to theXS52 DC cultures (5×10⁵ cells/ml). Culture supernatants collected 48 hrlater were examined for the indicated cytokines by ELISA. Results areexpressed as the means+/−s.e.m.from triplicate samples.

[0026] FIGS. 6A-C. Impact of Pep-1 on hapten-induced LC maturation.(FIG. 6A) Epidermal cells isolated from BALB/c mice at 17 hr aftertopical application of 0.5% DNFB or vehicle alone were double-stainedwith FITC-conjugated anti-IA mAb and PE-conjugated anti-CD86 mAb. Datashown are representative two-color FACS profiles, documenting markedelevated surface expression of CD86 by IA⁺ epidermal cells (i.e., LC)after DNFB application. (FIG. 6B) BALB/c mice received subcutaneous twolocal injections of Pep-1 or RP (40 μg/injection/ear) 24 and 3 hoursbefore topical application of 0.5% DNFB and epidermal cells wereisolated 17 hr later to examine the expression levels of CD86 on LC.Data shown are representative two-color FACS profiles, indicating thatDNFB-induced CD86 up-regulation in IA⁺ LC is prevented by locallyadministered Pep-1 . (FIG. 6C) Graphical representation of the % CD86⁺cells/IA⁺ LC from three independent experiments described in (FIG. 6B)with circles, triangles, and squares showing corresponding pairs in eachexperiment. Bars represent the mean values (n=3) and the asteriskindicates statistically significant difference between the Pep-1 and theRP panels (*p<0.03 by the Mann-Whitney U test).

[0027] FIGS. 7A-C. Impact of tetrameric LIP on cytokine productionduring antigen presentation. (FIG. 7A) XS52 DC (0.2×10⁶ cells/well) werecultured in the presence or absence of the KLH-reactive CD4⁺ Thl cloneHDK-1 (0.2×10⁶ cells/well) and/or KLH (100 μg/ml). Culture supernatantscollected at 24 hrs were examined for the indicated cytokines by ELISA.Data shown are the means+/−s.e.m. from triplicate samples. (FIG. 7B)XS52 DC and HDK-1 T cells were co-cultured with KLH in the presence ofthe indicated concentrations of Pep-1. Culture supernatants collected at24 hrs were examined for the indicated cytokines by ELISA. (FIG. 7C)CD4⁺ T cells isolated from DO11.10 transgenic mice (5×10⁴ cells/well)were co-cultured with the indicated numbers of splenic DC isolated fromBALB/c mice and the OVA peptide (2 μg/ml) in the presence of 500 μg/mlof Pep-1 (open circles), RP (triangles), or DMSO alone (closed circles).Co-cultures of CD4⁺ T cells and DC in the absence of OVA peptide servedas a control (square). All the cultures were examined for ³H-thymidineuptake on Day 3. Data shown are the means+/−s.d. from triplicatesamples.

[0028] FIGS. 8B-D. Synthesis and surface expression of HA by DC. (B)XS106 DC were labeled with ³H-glucosamine and then examined for theincorporation of hyaluronidase-sensitive radioactivities(means+/−s.e.m.; n=5) into the indicated subcellular fractions. (C)XS1006 DC were incubated with biotinylated Pep-1 or RP and then withPE-conjugated streptavidin (filed histograms) (Mummert et al. J.Exp.Med.192:769-779, 2000) or bovine testis hyaluronidase. Baseline staininglevels with PE-streptavidin alone are shown with open histograms. (D)The indicated DC preparations were examined for binding of biotinylatedPep-1 (filled) and RP (open). Peptide binding to splenic DC and bonemarrow-DC was examined within the CDllc⁺ populations. Binding ofbiotinylated Pep-1 was significantly diminished after hyaluronidasepretreatment in each DC preparation (data not shown).

[0029] FIGS. 9A-G. DC-associated HA and DC-dependent T cellproliferation. (A) DO11.10 T cells were cultured with y-irradiatedsplenic DC and/or OVA₃₂₃₋₃₃₉ (2 μg/ml) in the presence of intact bPG(250 μg/ml), heat-denatured bPG, or PBS alone. Data shown are themeans+/−s.d. (n=3) of ³H-thymidine uptake. (B) DO11.10 T cells werecultured with the indicated numbers of splenic DC and OVA₃₂₃₋₃₃₉ in thepresence of Pep-1 (300 μM) (open circles), RP (closed circles), or PBSalone (squares). (C) Pep-1 (open circles) or PEG-conjugated Pep-1 (opentriangles) were compared at the indicated concentrations (in terms ofthe molarity of the 12-mer Pep-1 sequence) for their abilities toinhibit the adhesion of ³⁵S-labeled B16 melanoma cells onto theHA-coated plates (Mummert et al. J.Exp.Med. 192:769-779, 2000). The %cell adhesion to non-coated plates is indicated with an open square. RP(closed circle) and PEG-conjugated RP (closed triangle) served ascontrols. (D) The indicated inhibitors (300 μM) were compared for theirabilities to inhibit the binding of FITC-labeled HA to BW5147 cellsurfaces (filled histograms) (Mummert et al. J.Exp.Med. 192:769-779,2000). Maximal binding in the absence of added inhibitors andautofluorescence in the absence of FITC-HA are indicated with solidlines and broken lines, respectively. (E) Pep-1 (open circles) orPEG-conjugated Pep-1 (open triangles) were compared at the indicatedconcentrations for their abilities to inhibit DO11.10 T cellproliferation triggered by splenic DC. (F) DO11.10 T cells were culturedwith the indicated numbers of bone marrow-derived DC and OVA₃₂₃₋₃₃₉ inthe presence of PEG-Pep-1 (60 μM) (open triangles), PEG-RP (closedtriangles), or PBS alone (open square). The baseline response in theabsence of OVA₃₂₃₋₃₃₉ is indicated with a closed square. (G) DO11.10 Tcells were cultured with splenic DC and OVA₃₂₃₋₃₃₉ in the presence ofanti-CD44 mAb (KM81) (open circles) or isotype-matched control IgG(closed circle) at the indicated concentrations. The same anti-CD44 mAbpreparation was examined in parallel for its ability to inhibit theadhesion of ³⁵S-labeled T cells onto the HA-coated plates (opentriangles). All the data in this figure are representative of at leasttwo independent experiments. Statistically significant differencesassessed by a two-tailed Student's t-test for comparing two groups or byanalysis of variance (ANOVA) followed by LSD for multiple comparisonsare indicated by asterisks (*P<0.05, **P<0.01).

[0030] FIGS. 10A-B. DC-associated HA mediates DC-T cell communicationduring antigen presentation. (A) CSFE-labeled DO11.10 T cells werecultured with splenic DC and/or OVA₃₂₃₋₃₃₉ in the presence of PEG-Pep-1(60 μM), PEG-RP, or PBS alone. Data shown are the CSFE fluorescenceprofiles within the PI-negative populations. (B) DO11.10 T cells werecultured with splenic DC and/or OVA₃₂₃₋₃₃₉ in the presence of PEG-Pep-1(60 μM), PEG-RP, or PBS alone. Culture supernatants (24 hr) were thentested for the indicated cytokines by ELISA. All data shown in thisfigure are representative of two independent experiments.

[0031]FIG. 11.A-B. In vivo inhibition of DC-dependent T cell activationby PEG-Pep-1. (A and B) CSFE-labeled DO11.10 T cells (10⁶ cells/animal)and OVA₃₂₃₋₃₃₉-pulsed bone marrow-DC (3×10⁵ cells/animal) were injectedintraperitoneally into BALB/c mice. These animals also receivedsimultaneous intraperitoneal injection of PEG-Pep-1 (20 μg/animal),PEG-RP, or PBS alone. Data shown are representative CSFE fluorescenceprofiles and % dividing cells within the PI-negative populationscollected from the peritoneal cavity 48 hr later (A) and themeans+/−s.e.m. of % dividing cells from triplicate animals (B). Datashown in this figure are representative of two independent animalexperiments.

DESCRIPTION OF ILLUSTRATIVE EMBODIMENTS

[0032] Agents that bind glycosaminoglycans such as chondroitin sulfate,chondroitin sulfate C, dermatan sulfate, heparin, keratan sulfate,keratosulfate, chitin, chitosan 1, chitosan 2 and especially hyaluronicacid (HA) can be useful therapeutic agents given the vast number ofphysiological processes and pathologies that involve glycosaminoglycans.The present inventors have previously isolated, by phage displaytechnology, peptides that bind to glycosaminoglycans (see co-pendingU.S. Provisional Serial No. 60/126,475, incorporated herein in itsentirety by reference; and Mummert et al. J.Exp.Med. 192:769-779, 2000).All references, to the extent that they provide exemplary procedural orother details supplementary to those set forth herein, are specificallyincorporated herein by reference in their entirety.

[0033] In one example, co-pending U.S. Provisional Serial No. 60/126,475describes, the development of a novel peptide that bindsglycosaminoglycans, termed “Pep-1 ,” by using phage display technology.This peptide has the sequence GAHWQFNALTVR. Pep-1 showed specificbinding to soluble, immobilized, and cell-associated forms of HA, andinhibited leukocyte adhesion to HA substrates almost completely.Systemic, local, or topical administration of Pep-1 inhibited theexpression of contact hypersensitivity responses in mice by blockingskin-directed homing of inflammatory leukocytes. Pep-1 also inhibitedthe sensitization phase by blocking hapten-triggered migration ofLangerhans cells (LCs) from the epidermis. These observations documentthat HA plays an essential role in “two-way” trafficking of leukocytesto and from an inflamed tissue, and thus provides HA inhibitors astherapeutic agents for inflammatory disorders.

[0034] The present invention provides derivatives of peptides that bindto glycosaminoglycans with a binding affinity K_(a) of 5×10⁵ l/mol ormore relative to a naturally occurring glycosaminoglycan in situ, andfurther shows that the invention the molecules can be used to manipulatemany aspects of the immune system, such as cytokine release, antigenpresentation, clonal expansion of T-cells, maturation ofantigen-presenting cells (APCs) such as dendritic cells, APC-T cellcluster formation, T-cells activation, cell adhesion and leucocyteextravasation. The invention can also be used to manipulatetumorigenesis, particularly metastasis. The derivatives includemultimers and/or chemically modified derivatives that have surprisinglyimproved biological, chemical, and physical properties. For example,multimeric Pep-1 derivatives, especially a Pep-1 dimer and a Pep-1tetramer, that have greater solubility, stability, and biologicalactivity as compared to Pep-1 are provided. It is envisioned that avariety of different multimers, in addition to those exemplified herein,will exhibit improved properties as compared to monomers, and may evenprovide synergistic improvement as compared to a comparable number ofmonomers.

[0035] The artificial peptide multimers of the invention preferably bindglycosaminoglycans with a binding affinity of at least K_(a) of 5×10⁵l/mol of more, preferably a K_(a) of 1×10⁶ l/mol or more, morepreferably a K_(a) of 1×10⁷ l/mol or more, or even more preferably aK_(a) of 1×10⁸ l/mol or more, 1×10⁹ l/mol or more, 1×10¹⁰ l/mol or more,1×10¹¹ l/mol or more in vivo, in vitro, in situ, or under standardlaboratory experimental conditions for measuring binding affinity.Binding may be to any gycosaminoglycan, preferably hyaluronic acid.

[0036] The invention also provides methods for preparing derivatives ofthe peptide-inhibitors of glycosaminoglycans. In one embodiment, Pep-1multimerization can be accomplished by methods that involve succinicacid bridging, PEG-conjugation, use of various linkers or linkingagents, as well as using other reagents that can bridge or chemicallymediate multimer formation. For example, a Pep-1 dimer was prepared bysuccinic acid bridging and a Pep-1 tetramer was prepared byPEG-conjugation. In addition to the dimer and the tetramer, theinventors contemplate that multimers including, trimers, pentamers,hexamers, etc., of Pep-1 also will have potent biological activities.The peptide multimer derivatives of the invention are contemplated tocomprise two or more peptide subunits, each subunit comprising between 6and 30 amino acids. In addition, the inventors also contemplate that themultimers can comprise both identical as well as heterogeneous peptidesubunits.

[0037] Also provided are derivatives of the peptides of the inventionthat are obtained by chemical modifications. For example, the peptidescan be amidated, PEGylated, acetylation, glycosylated, phosphorylated,carboxylation, carbonylation, or extended by attaching extensionsequence. In one embodiment, the extension sequence comprises arginineresidues. Other types of extension sequences are also contemplated. Insome embodiments the extension sequences may be a linker sequence. It isalso contemplated that in some cases the peptides of the invention willbe derivatized by one or more of the various chemical modificationmethods and/or the multimerization methods.

[0038] The present inventors have also demonstrates anti-cancerproperties of the peptide-inhibitors of glycosaminoglycans. For example,the Pep-1 monomer inhibited lung metastasis of melanoma. Thus, theinvention envisions anti-cancer therapies, especially those aimed atprevention of tumor metastasis, using peptide-inhibitors ofglycosaminoglycans including Pep-1 and its other chemical derivatives.The invention also envisions the use of monomer and tetramerpeptide-inhibitors of glycosaminoglycans in cancers involving increasedglycosaminoglycans or other glycosaminoglycan related-events.

[0039] The peptide-inhibitors of glycosaminoglycans also inhibitinteractions of antigen presenting cells with T-cells. For example, thePep-1 monomer and the Pep-1 tetramer inhibit interaction of dendriticcells (DC) with T cell in vitro. More specifically, Pep-1 and itsderivatives inhibit DC-dependent activation of naive T cells as measuredby the proliferative responses of CD4⁺ T-cells freshly isolated fromDO11.10 transgenic mice to splenic DC pulsed with ovalbumin (OVA)peptide. Pep-1 and its derivatives also interfere with antigenpresentation to memory T cells as seen in experiments with a HDK-1T-cell clone. These results demonstrate that Pep-1 and its derivativessuppress immune responses not only by blocking HA-dependent, two-waytrafficking of leukocytes but also by interfering with DC-dependentpresentation of antigens to both naive T-cells (DO11.10) and memoryT-cells (HDK-1 T cell clone). In addition, the invention demonstratesthat Pep-1 and its derivatives inhibit the secretion of cytokines suchas interferon-γ, IL-6, and TNFα in a co-culture system comprising of theXS52 DC line, the HDK-1 T-cell clone, and an antigen, such as thekeyhole limpet hemocyanin (KLH) antigen.

[0040] Thus, in light of the above, the peptide inhibitors ofglycosaminoglycans, including Pepl and its derivatives, are involved invarious aspects of immune system function including, the control ofactivation, differentiation, proliferation, trafficking, release ofcytokines and other regulatory/effector molecules by immune cells.Therefore, it is contemplated that the peptide inhibitors ofgylcosaminoglycans will be effective therapeutic agents in a variety ofimmune disorders. For example, Pep-1 and its derivatives are potentanti-inflammatory molecules by their ability to suppress immune systemand more specifically by their ability to prevent leukocyte trafficking.Preventing leukocyte trafficking to and from sites of inflammationprevents the associated tissue damage.

[0041] Over the last a few years, there has been accumulating evidenceto support the new concept that degradation products of HA (i.e., HAfragments), just like fragments of other extracellular matrix components(e.g., collagens and fibronectin), act as pro-inflammatory mediators.The present inventors have demonstrated that HA fragments trigger IL-6and TNFα production by the XS52 DC line. In addition, the inventors havedemonstrated that the glycosaminoglycan peptide-inhibitors of theinvention, including Pep-1 and its derivatives, significantly block theproduction of both cytokines by XS52 cells following stimulation with HAfragments. Thus, Pep-1 and its derivatives demonstrate pharmacologicalactivities that suppresses immune responses. The inventors thereforecontemplate methods for treatment of immune disorders such asinflammatory diseases, autoimmune diseases, and transplantation relatedgraft versus host diseases by administering to a patient in need thereofPep-1 and its derivatives as well as other glycosaminoglycan inhibitors.Also contemplated are methods for analyzing the biological activities ofHA fragments and HA metabolism utilizing the DC lines.

[0042] The inventors also have detected, using RT-PCR methods, mRNA forhyaluronidases and for hyaluronan synthases in keratinocytes. Severalenzymes involved in HA metabolism were found to be expressed by the Pam212 keratinocyte line and in mouse skin. This implies that keratinocytesand other skin cells can potentially produce HA fragments in response toenvironmental stimuli. Thus, the inventors envision pharmaceuticalcompositions of glycosaminoglycan peptide-inhibitor conjugates inventionfor topical therapeutic application to neutralize the pro-inflammatoryactivities of HA and locally produced HA fragments. Hyaluronases are oneclass of inhibitor that may be used in this context. Other agents thatcause HA degradation can be identified using standard screening assays.

[0043] Furthermore, the inventors have shown that HA is involved in Tcell communication and proliferation and is able to activate naive Tcells. These effects can be inhibited by the molecules of the invention,demonstrating that the molecules of the invention can be utilized toinhibit a wide range of gylcosaminoglycan-related reactions.

[0044] A more detailed discussion of these embodiments is provided inthe following pages.

[0045] A. Peptides

[0046] The peptides of the invention comprise those peptides that bindto glycosaminoglycans, or fragments thereof, and thereby modulate theirfunction. The peptides are contemplated to comprise between 6 and 30amino acid residues, and include multimeric derivatives as well as thoseformed by chemical modifications. The invention also encompassesnon-peptide analogs of the peptides, such as mimetics. Such derivativesand analogs are functionally active, i.e., capable of inhibiting one ormore functions associated glycosaminoglycan signaling. For example,peptides, derivatives or analogs can reduce immune reaction,prevent/reduce an inflammatory reaction, inhibit antigen presentingcell-T cell interaction, inhibit leukocyte trafficking, and inhibittumor metastasis.

[0047] In addition, derivatives of the peptides of the invention can bechemically synthesized using of a peptide synthesizer and standardsynthetic procedures. For example, peptides can be synthesized on asolid support (resin) using either Boc or Fmoc chemistries. Any type ofautomated synthesizer may be used including batch synthesizers and acontinuos-flow synthesizers. Alternatively, a manual synthetic approachmay be used to prepare the peptides of the invention. Alternatively,solution phase peptide synthesis methods can also be used.

[0048] In one embodiment, the peptide will have the core sequence ofgeneral formula (Z)_(n)X(Y)_(m), where X is any amino acid and each of Yand Z is an aliphatic or polar aliphatic amino acid, X is any amino acidand n and m are 6 to 30. A more specific embodiment is GAXWQFXALTVX,wherein X is any amino acid. Additions to these sequences can compriseextension sequences at either the C-terminal or N-terminal ends. In oneembodiment, this extension sequence is a C-terminal extension of W_(p)where W is any basic or neutral amino acids, and p is 3-13. In anotherembodiment the extension sequence is made of glycine repeats. In aspecific embodiment, the sequence is GAHWQFNALTVR or GAHWQFNALTVR. Inparticular embodiments, the R residue may be substituted by withalanine. Further embodiments subunits comprising the sequence of SEQ IDNO 16, or SEQ ID NO: 17.

[0049] The invention also contemplates, peptide derivatives that can bemade by altering the core sequences by substitutions, additions ordeletions that provide for functionally equivalent molecules. Forexample, some substitution derivatives contemplated include, but are notlimited to, those containing, as a primary amino acid sequence, all orpart of the amino acid sequence of the peptide with one or more aminoacid residues substituted by another amino acid of a similar polaritywhich acts as a functional equivalent, resulting in a silent alteration.

[0050] Substitutes for an amino acid within the sequence may be selectedfrom other members of the class to which the amino acid belongs. Forexample, the nonpolar or hydrophobic amino acids include alanine,leucine, isoleucine, valine, proline, phenylalanine, tryptophan andmethionine. Polar neutral amino acids include glycine, serine,threonine, cysteine, tyrosine, asparagine, and glutamine. Positivelycharged or basic amino acids include arginine, lysine and histidine andnegatively charged or acidic amino acids include aspartic acid andglutamic acid.

[0051] Furthermore, if desired, non-classical amino acids or chemicalamino acid analogs can be introduced as a substitution or addition intothe peptide sequence. Non-classical amino acids include but are notlimited to the D-isomers of the common amino acids, α-amino isobutyricacid, 4-amino-butyric acid, 2-amino butyric acid, γ-amino butyric acid,ε-6-amino hexanoic acid, 2-amino isobutyric acid, 3-amino propionicacid, omithine, norleucine, norvaline, hydroxyproline, sarcosine,citrulline, cysteic acid, t-butylglycine, t-butylalanine, phenylglycine,cyclohexylalanine, β-alanine, fluoro-amino acids, designer amino acidssuch as β-methyl amino acids, Cα-methyl amino acids, Nα-methyl aminoacids, and amino acid analogs in general. Furthermore, the amino acidcan be either of the optical isomers, D (dextrorotary) or L(levorotary).

[0052] The peptide derivatives and analogs of the invention can beproduced by various methods known in the art. Typically the basicpeptide will be synthesized as the size of the peptides is suitable forartificial synthesis. However, one may also obtain the core peptidesequence by recombinant DNA methods by any of numerous strategies knownin the art (Sambrook et al. Molecular Cloning: A Laboratory Manual, 2ndEd. (1989) Cold Spring Harbor Press, Cold Spring Harbor, N.Y.). Thesequence can be cleaved at appropriate sites with restrictionendonuclease(s), followed by further enzymatic modification if desired,isolated, and ligated in vitro. This product can then be subjected tochemical or enzymatic derivatization methods.

[0053] Chemical modification of the peptide sequences includeglycosylation, acetylation (including N-terminal acetylation),carboxylation, carbonylation, phosphorylation, PEGylation, amidation,use of non-peptide bonding (e.g., trans olefins), substitution ofα-hydrogens with methyl groups, derivatization by knownprotecting/blocking groups, circularization, inhibition of proteolyticcleavage (e.g., using D amino acids), linkage to an antibody molecule orother cellular ligand, etc. Any of numerous chemical modifications maybe carried out by known techniques, including but not limited tospecific chemical cleavage by cyanogen bromide, trypsin, chymotrypsin,papain, V8 protease, NaBH₄, acetylation, formylation, oxidation,reduction, etc.

[0054] As used herein “PEP 1” encompasses polypeptides having sequencesimilarity or sequence identity to SEQ ID NO: 1 and polypeptide relatedto SEQ ID NO: 1, such as SEQ ID NO: 2, polypeptides comprising thesequence (Z)_(n)X(Y)_(m), wherein X is any naturally occurring aminoacid and Z and Y are each independently selected from the groupconsisting of aliphatic amino acids and polar aliphatic amino acids, nand m are each independently an integer in the range of from 6 to 30,and wherin the peptide multimer has a binding affinity K_(a) of 5×10⁵l/mol or more relative to a naturally occurring glycosaminoglycan insitu. PEP 1 also encompases polypeptides of the formula ZZZXZZZ whereinZ is an amino acid selected from the group consisting of aliphatic andpolar aliphatic residues, and wherein X is any amino acid. The subunitmay further comprises an N- or C-terminal extension W_(p), wherein W isany basic or neutral amino acid, and p is an integer between 3 and 13,for example, wherein W is arginine or glycine independently. The peptidesubunit also may comprise a terminal serine. In a particular embodiment,the extension is a C-terminal GGGS. PEP 1 can have of at least about65%, preferably at least about 80%, more preferably at least about 85%,and can be about 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99% ormore identity to SEQ ID NO: 1 or 2. Sequence similarity and sequenceidentity are calculated based on a reference sequence, which will be atleast about 6 amino acids long, more usually at least about 10 aminoacids long, and may extend to the complete sequence that is beingcompared. In general, percent sequence identity is calculated bycounting the number of residue matches (e.g., nucleotide residue oramino acid residue) between the query and test sequence and dividingtotal number of matches by the number of residues of the individualsequences found in the region of strongest alignment. Thus, where 10residues of an 11 residue query sequence matches a test sequence, thepercent identity above would be 10 divided by 11, or approximately,90.9%. Algorithms for computer-based sequence analysis are known in theart, such as BLAST (see, e.g., Altschul et al., J. Mol. Biol.,215:403-10 (1990)), particularly the Smith-Waterman homology searchalgorithm as implemented in MPSRCH program (Oxford Molecular). For thepurposes of this invention, a preferred method of calculating percentidentity is the Smith-Waterman algorithm, using the following. GlobalDNA sequence identity must be greater than 65% as determined by theSmith-Waterman homology search algorithm as implemented in MPSRCHprogram (Oxford Molecular) using an affine gap search with the followingsearch parameters: gap open penalty, 12; and gap extension penalty, 1.

[0055] 1. Linkers/Coupling Agents/Extensions

[0056] In preparing peptide multimers of the present invention, one mayuse a variety of linking or coupling agents. For example, one can joinindividual peptide subunits to form multimers using abiologically-releasable bond, such as a selectively-cleavable linker oramino acid sequence. For example, peptide linkers that include acleavage site for an enzyme preferentially located or active within atumor environment are contemplated. Exemplary forms of such peptidelinkers are those that are cleaved by urokinase, plasmin, thrombin,Factor IXa, Factor Xa, or a metallaproteinase, such as collagenase,gelatinase, or stromelysin.

[0057] Additionally, while numerous types of disulfide-bond containinglinkers are known which can successfully be employed to conjugatemoieties, certain linkers will generally be preferred over otherlinkers, based on differing pharmacologic characteristics andcapabilities. For example, linkers that contain a disulfide bond that issterically “hindered” are to be preferred, due to their greaterstability in vivo, thus preventing release of the moiety prior tobinding at the site of action.

[0058] Additionally, any other linking/coupling agents and/or mechanismsknown to those of skill in the art can be used to combine the peptidesof the present invention, such as, for example, antibody-antigeninteraction, avidin biotin linkages, amide linkages, ester linkages,thioester linkages, ether linkages, thioether linkages, phosphoesterlinkages, phosphoramide linkages, anhydride linkages, disulfidelinkages, ionic and hydrophobic interactions, bispecific antibodies andantibody fragments, or combinations thereof.

[0059] Cross-linking reagents are used to form molecular bridges thattie together functional groups of two different molecules, e.g., astabilizing and coagulating agent. However, it is contemplated thatdimers or multimers of the same analog/protein subunit can be made orthat heteromeric complexes comprised of different analogs/proteinsubunits can be created. To link two different compounds in a step-wisemanner, hetero-bifunctional cross-linkers can be used that eliminateunwanted homopolymer formation. Table 1 provides a list ofhetero-bifunctional cross-linkers. TABLE 1 HETERO-BIFUNCTIONALCROSS-LINKERS Spacer Arm Length After Linker Reactive Toward Advantagesand Applications Cross-Linking SMPT Primary amines Greater stability11.2 A Sulfhydryls SPDP Primary amines Thiolation  6.8 A SulfhydrylsCleavable cross-linking LC-SPDP Primary amines Extended spacer arm 15.6A Sulfhydryls Sulfo-LC-SPDP Primary amines Extended spacer arm 15.6 ASulfhydryls Water-soluble SMCC Primary amines Stable maleimide reactivegroup 11.6 A Sulfhydryls Enzyme-antibody conjugation Hapten-carrierprotein conjugation Sulfo-SMCC Primary amines Stable maleimide reactivegroup 11.6 A Sulfhydryls Water-soluble Enzyme-antibody conjugation MBSPrimary amines Enzyme-antibody conjugation  9.9 A SulfhydrylsHapten-carrier protein conjugation Sulfo-MBS Primary aminesWater-soluble  9.9 A Sulfhydryls SIAB Primary amines Enzyme-antibodyconjugation 10.6 A Sulfhydryls Sulfo-SIAB Primary amines Water-soluble10.6 A Sulfhydryls SMPB Primary amines Extended spacer arm 14.5 ASulfhydryls Enzyme-antibody conjugation Sulfo-SMPB Primary aminesExtended spacer arm 14.5 A Sulfhydryls Water-soluble EDC/Sulfo-NHSPrimary amines Hapten-Carrier conjugation 0 Carboxyl groups ABHCarbohydrates Reacts with sugar groups 11.9 A Nonselective

[0060] An exemplary hetero-bifunctional cross-linker contains tworeactive groups: one reacting with primary amine group (e.g., N-hydroxysuccinimide) and the other reacting with a thiol group (e.g., pyridyldisulfide, maleimides, halogens, etc.). Through the primary aminereactive group, the cross-linker may react with the lysine residue(s) ofone protein (e.g., the selected antibody or fragment) and through thethiol reactive group, the cross-linker, already tied up to the firstprotein, reacts with the cysteine residue (free sulfhydryl group) of theother protein (e.g., the selective agent).

[0061] It is preferred that a cross-linker having reasonable stabilityin blood will be employed. Numerous types of disulfide-bond containinglinkers are known that can be successfully employed to conjugatetargeting and therapeutic/preventative agents. Linkers that contain adisulfide bond that is sterically hindered may prove to give greaterstability in vivo, preventing release of the targeting peptide prior toreaching the site of action. These linkers are thus one group of linkingagents.

[0062] Another cross-linking reagent is SMPT, which is a bifunctionalcross-linker containing a disulfide bond that is “sterically hindered”by an adjacent benzene ring and methyl groups. It is believed thatsteric hindrance of the disulfide bond serves a function of protectingthe bond from attack by thiolate anions such as glutathione which can bepresent in tissues and blood, and thereby help in preventing decouplingof the conjugate prior to the delivery of the attached agent to thetarget site.

[0063] The SMPT cross-linking reagent, as with many other knowncross-linking reagents, lends the ability to cross-link functionalgroups such as the SH of cysteine or primary amines (e.g., the epsilonamino group of lysine). Another possible type of cross-linker includesthe hetero-bifunctional photoreactive phenylazides containing acleavable disulfide bond such as sulfosuccinimidyl-2-(p-azidosalicylamido) ethyl-1,3′-dithiopropionate. The N-hydroxy-succinimidylgroup reacts with primary amino groups and the phenylazide (uponphotolysis) reacts non-selectively with any amino acid residue.

[0064] In addition to hindered cross-linkers, non-hindered linkers alsocan be employed in accordance herewith. Other useful cross-linkers, notconsidered to contain or generate a protected disulfide, include SATA,SPDP and 2-iminothiolane (Wawrzynczak & Thorpe, 1987). The use of suchcross-linkers is well understood in the art. Another embodiment involvesthe use of flexible linkers.

[0065] U.S. Pat. No. 4,680,338, describes bifunctional linkers usefulfor producing conjugates of ligands with amine-containing polymersand/or proteins, especially for forming antibody conjugates withchelators, drugs, enzymes, detectable labels and the like. U.S. Pat.Nos. 5,141,648 and 5,563,250 disclose cleavable conjugates containing alabile bond that is cleavable under a variety of mild conditions. Thislinker is particularly useful in that the agent of interest may bebonded directly to the linker, with cleavage resulting in release of theactive agent. Preferred uses include adding a free amino or freesulfhydryl group to a protein, such as an antibody, or a drug.

[0066] U.S. Pat. No. 5,856,456 provides peptide linkers for use inconnecting polypeptide constituents to make fusion proteins, e.g.,single chain antibodies. The linker is up to about 50 amino acids inlength, contains at least one occurrence of a charged amino acid(preferably arginine or lysine) followed by a proline, and ischaracterized by greater stability and reduced aggregation. U.S. Pat.No. 5,880,270 discloses aminooxy-containing linkers useful in a varietyof immunodiagnostic and separative techniques.

[0067] These other cross-linking agents can also be used to linkextension sequences to the peptides of the invention. For example, onecan link an extension peptide sequence to the terminal ends of thepeptides of the invention, i.e., at the C-terminal end and/or at theN-terminal end. Thus, any peptide sequence, or motif can be linked tothe peptides of the invention using a suitable linking agent.

[0068] 2. Chemical Modifications

[0069] Other methods for obtaining the peptide derivatives of theinvention involve chemical modifications. The peptide compositions canbe modified by amidation, PEGylation, glycosylation, acetylation,prenylation, phosphorylation, biotinylation, carboxylation,carbonylation, or may be further derivatized by addition of knownprotecting/blocking groups. These chemical modifications can be made bymethods well known to the skilled artisan. Alternatively, one can alsouse enzymes to achieve such modifications. For example, any kinaseenzyme can be used for phosphorylation, specifically serine-threoninekinase enzymes phosphorylate serine or threonine residues and tyrosinekinases may be used to phosphorylate tyrosine residues in the peptide.

[0070] One can also derivatize peptides by adding or attaching anon-peptide ligand. The ligand may comprise of, but is not limited to,an acetamido, an acetoacetamido, an acetoacetyl, an acetonyl, anacetonylidene, an acetyl, an acrlyl, an adipyl, an alanyl, abeta-alanyl, an allophanoyl, an allyl, an allylidene, an amidino, anamino, an amyl, an anilino, an anisidino, an anisyl, an anthranoyl, anarsino, an azelaoyl, an azido, an azino, an azo, an azoxy, a benzal, abenzamido, a benzhydryl, a benzimido, a benzoxy, a benzoyl, a benzyl, abenzlidine, a benzyldyne, a biphenylyl, a biphenylene, a bromo, abutoxy, a sec-butoxy, a tert-butoxy, a butyl, an iso-butyl, a sec-butyl,a tert-butyl, a butyryl, a caproyl, a capryl, a caprylyl, a cabamido, acabamoyl, a cabamyl, a cabozoyl, a carbethoxy, a cabobenzoxy, acarbonyl, a carboxy, a cetyl, a chloro, a chloroformyl, a cinnamyl, acinnamoyl, a cinnamylidene, a cresyl, a crotoxyl, a crotyl, a cynamido,a cyanato, a cyano, a decanediol, a decanoly, a diazo, a diazoamine, adisilanyl, a disiloxanoxy, a disulfinyl, a dithio, an enanthyl, anepoxy, an ethenyl, an epoxy, an ethenyl, an ethinyl, an ethyl, anethylthio, a fluoro, a fluorenyl, a formamido, a formyl, a fumaroy, afurfuryl, a furfurylidene, a furyl, a glutamyl, a gularyl, a glycidyl, aglycinamido, a glycolyl, a glycyl, a glyoxylyl, a guanidino, a guanyl, aheptadecanoyl, a heptanamido, a heptanedioyl, a heptanoyl, ahexadecanoyl, a hexamethylene, a hexanedioyl, a hippuryl, a hydantoyl, ahyrazino, a hydrazo, a hydrocinnamoyl, a hydroperoxy, a hydroxamino, ahydroxy, an imino, an indenyl, an iodoso, an isoamyl, an isobutenyl, anisobutoxy, an isobutyl, an isobutylidene, an isobutyryl, an isocyanato,an isocyano, an isohexyl, an isoleucyl, an isonitroso, an isopentyl, anisopentylidene, an sopropenyl, an isopropoxy, an isopropl, anisopropylidene, an isothiocyanato, an isovaleryl, an iodo, a keto, alactyl, a lauroyl, a leucyl, a levulinyl, a malonyl, a mandelyl, amercapto, a methacrylyl, a methallyl, a methionyl, a methoxy, a methyl,a methylene, a methylenedioxy, a methylenedisulfonyl, a methylol, amethylthio, a myristyl, a naphthal, a naphthobenzyl, a naphthoxy, anaphthyl, a naphthylidene, a neopentyl, a nitramino, a nitro, anitrosamino, a nitrosimino, a nitroso, a nonanoyl, an oleyl, an oxalyl,an oxamindo, an oxo, a palmityl, a pelargonyl, a pentamethylene, apentyl, a phenacyl, a phenacylidene, a phenanthryl, a phenethyl, aphenoxy, a phenyl, a phenylene, a phenylenedioxy, a phosphino, aphosphinyl, a phospho, a phosphono, a phthalyl, a picryl, a pimely, apiperdino, a pieridyl, a pipemoyl, a pivalyl, a prenyl, a propargyl, apropenyl, a iso-propenyl, a propionyl, a propoxy, a propyl, aiso-propyl, a propylidene, a pyridino, a pyrryl, a salicyl, a selenyl, aseryl, a siloxy, a silyl, a silylene, a sorbyl, a stearyl, a styryl, asuberyl, a succinamyl, a succinyl, a sulfamino, a sulfamyl, asulfanilyl, a sulfeno, a sulfhydryl, a sulfinyl, a sulfo, a sulfonyl, aterephthalyl, a tetramethylene, a thenyl, a thienyl, a thiobenzoyl, athiocarbamyl, a thiocarbonyl, a thiocarboxy, a thiocyanato, a thionyl, athiophenacyl, a thiuram, a thronyl, a toluidino, a toluyl, a tolyl, aalpha-tolyl, a tolylene, a alpha-tolylene, a tosyl, a triazano, atrimethylene, a triphenylmethyl, a tyrosyl, a ureido, a valeryl, avalyl, a vinyl, a vinylidene, a xenyl, a xylidino, a xylyl, or axylylene ligand.

[0071] B. Glycosaminoglycan Function and Related Disease States

[0072] As glycosaminoglycans are involved in interactions with variouscellular receptors, agents that bind and modulate glycosaminoglycans areuseful as therapeutic agents in numerous diseases. For example, one ofthe cellular receptors of HA is CD44 is which is a family ofcell-surface glycoproteins generated by alternative splicing andpost-translational modification (Aruffo et al. Cell. 61:1303-1313, 1990;Lesley et al. Exp. Cell. Res. 187:224, 1990; Miyake et al. J.Exp.Med.172:69-75, 1990; Culty et al., J. Cell. Biol. 111:2765 1990). Variousisoforms of CD44 are expressed by different cell types, such as T cellsand B cells, granulocytes, monocytes, macrophages, and DC, including LC.HA-CD44 interaction mediates rolling of leukocytes over endothelialcells, one of the numerous processes that take place in leukocyte homing(DeGrendele et al. J.Exp.Med. 183:1119-1130, 1996; Clark et al. et al.J. Cell. Bio. 134:1075-1087, 1996). HA-CD44 interaction also is known tomediate many physiological and pathological events, including leukocytehoming, tumor metastasis, tissue development, hematopoiesis, cytokineproduction, T cell activation, and apoptosis.

[0073] Thus, pharmacological formulations of the peptide derivatives ofthe present invention can be used as therapeutic agents that modulateHA-CD44 interaction, and thus are useful to prevent and/or treatpatients with diseases mediated by this interaction. The nature of themodulation may comprise either inhibition or enhancement of theinteraction. Some of the diseases that the peptide derivatives providedhere are contemplated to be useful in include, contact hypersensitivityand delayed type hypersensitivity reactions (Camp et al. J.Exp.Med.178:497-507, 1993; Verdrengh et al. Scand. J Immunol. 42:353, 1995),tumor metastasis (Bartolazzi et al. J. Exp. Med. 180:53-66 1994; Guo etal., Cance.r Res. 54:1561 1994; Zahalka et al. J.Immunol.154:5345-5355,1995), collagen-induced autoimmune arthritis models(Verdrengh et al. Scand. J. Immunol. 42:353, 1995; Mikecz et al. Nat.Med. 1:558, 1995; Zeidler et al. Autoimmunity. 21:245, 1995). Inrelation to prevention of tumor metastasis, the present inventors haveshown that the peptides of the invention, particularly Pep-1 and itsderivatives, inhibit metastasis in a mouse model of malignant cancer.

[0074] The present inventors also have previously demonstrated thatpeptide inhibitors of HA can be used to prevent the induction of immunereactions in the sensitization phase and to inhibit inflammatoryreactions in the elicitation phase. For example, Mohamadzadeh et al. J.Invest. Dermatol., 1999 demonstrates that keratinocytes expressrelatively large amounts of cell-surface HA, and the long-term LC line,XS106, exhibits significant rolling over in HA-coated plates as well asover confluent keratinocyte monolayers under physiological flowconditions. This XS106 cell rolling is blocked by soluble HA and localinjection of soluble HA into mouse skin inhibits almost completely LCemigration that is triggered by topical application of DNFB. Thus, thepeptides of the present invention are useful to inhibit a mechanism ofDC migration, for example, an interaction between HA (on keratinocytes)and CD44 (on activated LC).

[0075] In addition to CD44, HA has several other ligands including,RHAMM, aggrecan, versican, link protein, the LEC HA receptor,hyaluronection, inter-α-trypsin inhibitor-related proteins, BEHAB, CD38,CD54, and hyaluronidase (HAase). Therefore, the peptide derivatives thatbind glycosaminoglycans, provided herein, are envisioned to be useful inpotentiating or inhibiting glycosaminoglycan-mediated activities throughthese receptor molecules as well. The skilled artisan will recognizethat the peptide derivatives provided here will be useful as therapeuticagents in various pathologies and that the scope of the invention is notlimited to the diseases and conditions mentioned here.

[0076] C. HA Metabolism

[0077] Schiller et al. J.Biol.Chem. 218:139-145, 1955, first studied theregional turnover of HA in the skin over forty years ago. Usingradioactive tracers, these investigators showed that HA has aphysiological half-life of 2-4 days. More recently, Fraser and Laurent(1989), have shown that the bulk of HA is not degraded in the tissue oforigin (i.e., regional turnover) but rather is transported to theregional lymph nodes for degradation. HA that escapes degradation in thelymph nodes enters the circulation via the thoracic duct where it iscatabolized predominantly by the liver. Three enzymatic reactions areresponsible for HA degradation, namely, a) hyaluronidase (HAase), b)β-D-glucuronidase, and c) β-N-acetyl-D-hexosaminidase (Roden et al.Ciba. Found.Symp. 143:60-76, 1989). Three unique HAases have beenidentified in humans to date (Lepperdinger et al. J.Biol.Chem.273:22466-22470, 1998; Gmachl et al. F.E.B.S. Lett. 336:545-548, 1993;).HAases degrades HA polysaccharides to oligosaccharides whileβ-D-glucuronidase and β-N-acetyl-D-hexosaminidase degradesoligosaccharides into monosaccharides (glucuronic acid andN-acetyglucosamine). Glucuronic acid is converted through a series ofenzymatic reactions to D-glucose-6-phosphate. The D-glucose-6-phosphatethen funnels into the glycolytic pathway. N-acetyglucosamine isphosphorylated in an ATP-dependent reaction to yieldN-acetyglucosamine-6-phosphate. The N-acetyglucosamine-6-phosphate isconverted to either UDP-N-acetyglucosamine or fructose-6-phosphate.UDP-N-acetyglucosamine serves as starting material for the synthesis ofnew polysaccharides while fructose-6-phosphate enters the glycolyticpathway (Roden et al. Ciba. Found. Symp. 143:60-76, 1989).

[0078] Stimuli that directly modulate the enzymatic degradation ofhyaluronan remain to be determined. However, Elias and colleagues(Sampson et al. J.Clin.Invest. 90:1492-1503, 1992) have shown acorrelation between the level of CD44 mRNA and HA degradation in humanlung fibroblasts after stimulation with recombinant IL-1 and TNF. Theseinvestigators speculated increased expression of CD44 may enhance HAbinding and result in increased HA uptake and consequent degradation.

[0079] Importantly products of HA catabolism have been associated withbiological functions. Recently, Noble and colleagues have shown that HAfragments, either alone or synergistically with cytokines, inducemacrophages to elaborate chemokines including MIP-1β, MIP-1α, Mig,interferon-inducible protein-10, RANTES, cytokine responsive gene-2 andmonocyte chemoattractant protein-1 (Horton et al. J.Biol.Chem.273:35088-35094, 1998; McKee et al. J.Clin.Invest. 98:2403-2413, 1996;Horton et al. J.Immunol. 160:3023-3030, 1998; Hodge-Dufour et al.J.Immunol. 159:2492-2500, 1997). HA fragments have also been shown to bepotent activators of DC and induce phenotypic maturation (up-regulationof MHC class II molecule, CD80, CD86, and CD83 and down-regulation ofCD115) and the production of IL-1β, TNFα and IL-12 (Termeer et al.J.Immunol. 165:1863-1870, 2000).

[0080] Synthesis of new HA polysaccharides is mediated by HA synthases.Like HAases, three synthases have been identified to date (Itano et al.Biochem. Biophys. Res.Commun. 222:816-820, 1996; Spicer et al.J.Biol.Chem., 271:23400-23406, 1996; Spicer et al. J.Biol.Chem.272:8957-8961, 1997; Watanabe et al. J.Biol.Chem. 271:22945-22948,1996).A variety of stimuli have been shown to initiate HA synthesis,including: a) environmental factors such as irradiation (Li et al.Am.J.Respir.Cell Mol. Biol. 23:411-418. 2000), culturing conditions(Huey et al. Matrix. 10:75-83, 1990) and hyperbaric oxygen (Roberts etal. Br.J.Dermatol. 131:630-633, 1994), b) HAase-mediated degradation ofHA from the plasma membrane surface (Philipson et al. Biochem.24:7899-7906, 1985; Larnier et al. Biochim.Biophys.Acta. 1014:145-152,1989) and, 3) various factors including IL-1β, TNFα, PDGF-AA, PDGF-BB,bFGF, EGF and TGF-β1 (Godden et al. Eur.J.Cancer. 35:473-480, 1999;Jacobson et al. Biochem.J. 348 Pt 1:29-35, 2000; Kennedy et al.J.Pediatr.Surg. 35:874-879, 2000; Ueki et al. Biochim.Biophys.Acta.1495:160-167, 2000; Denk et al. Curr.Eye. Res. 20:77-80, 2000).

[0081] The present inventors have demonstrated that keratinocytesexpress mRNA for HA synthase-2 and 3, as well as for HAases-1, 2, and 3,thus, showing that keratinocytes are fully capable of synthesizing anddegrading HA actively. The current inventors also have detected theexpression of these mRNAs in the skin, which indicates thatkeratinocytes (and other cell types in the skin, including DC) canpotentially produce HA fragments in response to environmental stimuli.

[0082] HA fragments have been shown to act as pro-inflammatorymediators. Additionally, the present inventors have demonstrated that HAfragments trigger IL-6 and TNFα production by the XS52 DC line. Theinventors have also demonstrated that the glycosaminoglycanpeptide-inhibitors of the invention, including Pep-1 and itsderivatives, significantly block the production of both cytokines byXS52 cells following stimulation with HA fragments. These observations,among others provide therapeutic methods utilizing the ability of thepeptides of the invention to neutralize the pro-inflammatory activitiesof locally produced HA fragments.

[0083] D. Therapeutic Uses

[0084] The peptide derivatives of the invention can be used astherapeutic and preventive agents for the treatment of various human andveterinary diseases associated with glycosaminoglycan function.Particular activities of the peptides include, but are not limited to,inhibiting leukocyte migration, inhibiting leukocyte activation,inhibiting cytokine production, inhibiting lymphocyte proliferation,inhibiting lymphocyte differentiation, inhibiting interaction betweencells (e.g., between an antigen presenting cell and a T cell),inhibiting dendritic cell migration, reduction of immune reaction,inhibition of dendritic cell migration, inhibition of metastasis, orbinding to a glycosaminoglycan and/or fragment thereof. In the contextof these applications, it is contemplated that the peptides of theinvention will be used either alone or in combination with othertherapeutic agents to accomplish these objectives.

[0085] Several of the peptide derivatives of the invention are solublein the absence of solvents and/or are also storage stable in water. Inaddition, several of the multimeric peptide derivatives in particularhave increased half-lives. These properties makes them extremelydesirable for therapeutic applications. However, in other instances itmay be desirable to further modify the peptides to provide these orother desirable characteristics for therapeutic use.

[0086] When used as a therapeutic, an appropriate dosage of thepharmaceutical formulation of the glycosaminoglycan binding agent, ormixture thereof, will be determined by any of several well establishedmethodologies. For instance, animal studies are commonly used todetermine the maximal tolerable dose, or MTD, of bioactive agent perkilogram weight. Other toxicity and pharmacokinetic profiles will alsobe determined. In general, at least one of the animal species tested ismammalian. Those skilled in the art regularly extrapolate doses forefficacy and avoiding toxicity to other species, including human.Additionally, therapeutic dosages may also be altered depending uponfactors such as the severity of infection, and the size or species ofthe host.

[0087] Preferably, animal hosts that may be treated using the peptidesof the present invention include, but are not limited to, invertebrates,vertebrates, birds, mammals such as pigs, goats, sheep, cows, dogs,cats, and particularly humans.

[0088] 1. Inflammatory Conditions

[0089] As the peptides of the invention are involved in numerous eventsthat lead to inflammatory responses, for e.g., inhibition of leukocytemigration, inhibition of leukocyte activation, inhibition of cytokineproduction, inhibition of lymphocyte proliferation, inhibition oflymphocyte differentiation, inhibition of interaction between an antigenpresenting cell and a T cell, and/or inhibition of dendritic cellmigration, etc. these peptides are envisioned to be useful in thetreatment and/or prevention of inflammation. For example, responses thatinhibit migration of leukocytes to and from sites of inflammation,prevent the associated cellular damage caused by the accumulation andactivation of leukocytes at these sites. Thus, patients suffering from avariety of inflammatory diseases including, infections, autoimmuneconditions, graft versus host diseases, etc. may be treated with acomposition of the invention.

[0090] Infections include all types of infections such as but notlimited to, viral infections, bacterial infections, fungal infections,burn infections, wound infections, etc. One important use of theglycosaminoglycan-specific peptide inhibitors of the invention is in thetreatment, amelioration and/or prevention of HIV infection. HIV andother infectious organisms are known to bind to HA (and otherglycosaminoglycans) upon infection of cells. Therefore, peptides of theinvention can be used to inhibit these interactions.

[0091] Autoimmune disease are exemplified by multiple sclerosis,rheumatoid arthritis, or systemic lupus erythematosus, etc., asnon-limiting examples. In addition, peptides of the invention may beuseful to prevent autoimmune inflammatory reactions stemming fromprocedures such as bone marrow and organ transplants, for example,suppression of graft-versus-host disease.

[0092] Administration of the anti-inflammatory composition of theinvention may be through a variety of routes. For example, one canadminister the composition systemically or locally or even topically,e.g., systemic treatment for lupus; injection into a joint to decreaseinflammation caused by arthritis; or topical application in the form ofa creme, ointment, balm, gel, etc. for an external topical infection.Alternately, where the targeted region of inflammation is internal,preparations of peptides may be provided by oral dosing. Additionally,pulmonary inflammation may be treated both parenterally and by directapplication of suitably formulated forms of the peptides to the lung byinhalation therapy or intranasal administration.

[0093] In addition to these inflammatory conditions, one may administera therapeutic anti-HA peptides and their derivatives to patientssuffering from a stroke or a myocardial infarction. In patientssuffering from a myocardial infarction the peptides of the invention canfacilitate a decrease in pressure upon myocardial tissues, preventtissue necrosis, and relieve edema (Maclean et al. Science. 194:199-200,1976; Opie et al. Am. Heart. J. 100:531-52, 1980). Use of peptideinhibitors of HA activity in treatment of myocardial infarct patients isadvantageous over use of therapeutic agents such as hyaluronidase(HAase) as the peptide's activity is specific to HA and thus will not beinhibited by heparin. Heparin is often administered during heart attacksand is a very powerful inhibitor of HAase activity of other HAasecontaining this EGF motif Because the peptide inhibitors of HA are notsubject to regulation by heparin, the clinician need not be concernedthat co-administration of heparin with the anti-HA peptide.

[0094] Peptide derivatives of the invention that inhibit HA activity canalso be used in the treatment of edema associated with brain tumors,particularly that associated with glioblastoma multiform. The edemaassociated with brain tumors results from the accumulation of HA in thenon-cancerous portions of the brain adjacent the tumor. Administrationof the HA-specific peptide inhibitor to the sites of hyaluronanaccumulation (e.g., by intravenous injection or via a shunt) can relievethe edema associated with such malignancies by binding to and preventingactivity of excess HA these sites. Thus, HA inhibitors can be successfulin the treatment of brain tumors not only in the reduction of the tumormass and inhibition of tumor growth and/or metastasis (see the sectioninfra for a discussion of the anti-cancer properties of the peptides ofthe invention), but it also is useful in relieving edema associated withthe malignancy.

[0095] 2. Combination Immunotherapies

[0096] The anti-inflammatory therapies comprising theglycosaminoglycan-binding inhibitors of the present invention can beused in conjunction with other therapies that are used for the treatmentof inflammation. Thus, one may use a peptide inhibitor of the inventionin combination with an anti-inflammatory agent. Anti-inflammatory agentsare agents that decrease the signs and symptoms of inflammation. A widevariety of anti-inflammatory agents are known to one of skill in theart. Most commonly used are the nonsteroidal anti-inflammatory agents(NSAIDs) which work by inhibiting the production of prostaglandins.Non-limiting examples include, ibuprofen, ketoprofen, piroxicam,naproxen, naproxen sodium, sulindac, celecoxib, aspirin, cholinesubsalicylate, diflunisal, oxaprozin, diclofenac sodium delayed release,diclofenac potassium immediate release, etodolac, ketorolac, fenoprofen,flurbiprofen, indomethacin, fenamates, meclofenamate, mefenamic acid,nabumetone, oxicam, piroxicam, salsalate, tolmetin, and magnesiumsalicylate. Another group of anti-inflammatory agents comprise steroidbased potent anti-inflammatory agents, for example, the corticosteroidswhich are exemplified by dexamethason, hydrocortisone,methylprednisolone, prednisone, and triamcinolone as non-limitingexamples. Several of these anti-inflammatory agents are available underwell known brand names, for example, the NSAIDs comprising ibuprofeninclude Advil, Motrin IB, Nuprin; NSAIDs comprising acetaminophensinclude Tylenol; NSAIDs comprising naproxen include Aleve.

[0097] It is conceivable that more than one administration of either theother anti-inflammatory agent and the peptide of the present inventionwill be required to achieve complete cure. Thus, various combinationsmay be employed, where the other anti-inflammatory agent is “A” and theanti-glycosaminoglycan peptide of the present invention is “B”, asexemplified below: A/B/A B/A/B B/B/A A/A/B B/A/A A/B/B B/B/B/A B/B/A/BA/A/B/B A/B/A/B A/B/B/A B/B/A/A B/A/B/A B/A/A/B B/B/B/A A/A/A/B B/A/A/AA/B/A/A A/A/B/A A/B/B/B B/A/B/B B/B/A/B

[0098] Other combinations also are contemplated.

[0099] 3. Cancer Therapies

[0100] The present inventors have shown that both theglycosaminoglycan-specific peptide inhibitors (e.g., Pep-1 and itsderivatives) as well as the glycosaminoglycan-specific peptidederivatives are useful in the treatment of cancer. Particularly,peptides of the invention, were shown to inhibit metastasis. It iscontemplated that the peptides of the invention will be used in thetreatment of brain cancer, lung cancer, throat cancer, esophagealcancer, cancer of the head and neck, skin cancer, breast cancer, stomachcancer, colon cancer, cancer of the rectum, cervical cancer, prostatecancer, ovarian cancer, liver cancer, pancreatic cancer, a cancer of theblood, small lung cell carcinoma, squamous lung cell carcinoma, as wellas any other cancer associated with increased levels of HA or otherglycosaminoglycans or of cancers that are associated with alteredglycosaminoglycan functions. It is also contemplated that the peptidesof the invention may increase the sensitivity of tumors that areresistant to conventional radiotherapy or chemotherapy. In addition, theinvention also contemplates the use of peptide mimetics.

[0101] Additionally, another important embodiment of the inventionprovides, for the first time, a successful anti-cancer effect byaltering glycosaminoglycan functions. Thus, the invention envisionsanticancer-therapies utilizing any agent that is capable of inhibitingglycosaminoglycan function or expression. Thus, provided are therapiesfor the treatment of cancers associated with increased levels of HA orother glycosaminoglycans, or of cancers that are associated with alteredglycosaminoglycan function, comprising providing to a patient in needthereof a agent that inhibits a glycosaminoglycan. Such an agent caninclude among others any peptide or non-peptide glycosaminoglycaninhibitor. Non-peptide inhibitors of the invention include naturallyoccurring products, including those isolated from any living organism;man-made products; chemical compounds; pharmaceutical compounds; peptidemimetics; small molecule inhibitors; compounds that may be designedthrough rational drug design starting from known inhibitors ofglycosaminoglycan; polynucleotides; antisense molecules; ribozymes;antibodies to glycosaminoglycans; etc.

[0102] The peptide and non-peptide glycosaminoglycan inhibitors of theinvention are contemplated to provide anti-cancer therapy either aloneor in combination with other anti-cancer agents. Severalcancer-therapies are currently prescribed for the treatment of cancers.Presented in sections below is a discussion of some of these therapeuticmethods.

[0103] 4. Combination Cancer Therapies

[0104] A wide variety of cancer therapies, known to one of skill in theart, may be used in combination with the anticancer peptides of thepresent invention. Thus, in order to increase the effectiveness of theanticancer therapy using a peptide of the present invention it may bedesirable to combine these compositions with other agents effective inthe treatment of cancer such as but not limited to those describedbelow.

[0105] For example, one can use the peptide inhibitors ofglycosaminoglycans of the invention for cancer therapy in conjunctionwith surgery and/or radiation therapy and/or chemotherapy, and/or genetherapy, and/or local heat therapy. All other non-peptide inhibitorsbased cancer therapies are referred to herein as “other cancertherapies.”

[0106] The other cancer therapy may precede or follow thepeptide-inhibitor-based therapy by intervals ranging from minutes todays to weeks. In embodiments where the other cancer therapy and thepeptide-inhibitor-based therapy are administered together, one wouldgenerally ensure that a significant period of time did not expirebetween the time of each delivery. In such instances, it is contemplatedthat one would administer to a patient both modalities within about12-24 hours of each other and, more preferably, within about 6-12 hoursof each other, with a delay time of only about 12 hours being mostpreferred. In some situations, it may be desirable to extend the timeperiod for treatment significantly, however, where several days (2, 3,4, 5, 6 or 7) to several weeks (1, 2, 3, 4, 5, 6, 7 or 8) lapse betweenthe respective administrations.

[0107] It also is conceivable that more than one administration ofeither the other cancer therapy and the peptide-inhibitor-based therapywill be required to achieve complete cancer cure. Various combinationsmay be employed, where the other cancer therapy is “A” and thepeptide-inhibitor-based therapy treatment is “B,” as exemplified below:A/B/A B/A/B B/B/A A/A/B B/A/A A/B/B B/B/B/A B/B/A/B A/A/B/B A/B/A/BA/B/B/A B/B/A/A B/A/B/A B/A/A/B B/B/B/A A/A/A/B B/A/A/A A/B/A/A A/A/B/AA/B/B/B B/A/B/B B/B/A/B

[0108] Other Combinations also are Contemplated.

[0109] In addition, the peptide-inhibitor-based therapy can beadministered to a patient in conjunction with other therapeutic methodssuch as for example standard AIDS treatments. The exact dosages andregimens can be suitable altered by those of ordinary skill in the art.Examples of cancer therapies are presented below.

[0110] a) Radiotherapeutic Agents

[0111] Radiotherapeutic agents and factors include radiation and wavesthat induce DNA damage for example, γ-irradiation, X-rays,UV-irradiation, microwaves, electronic emissions, radioisotopes, and thelike. Therapy may be achieved by irradiating the localized tumor sitewith the above described forms of radiation. Dosage ranges for X-raysrange from daily doses of 50 to 200 roentgens for prolonged periods oftime (3 to 4 weeks), to single doses of 2000 to 6000 roentgens. Dosageranges for radioisotopes vary widely, and depend on the half-life of theisotope, the strength and type of radiation emitted, and the uptake bythe neoplastic cells.

[0112] b) Surgery

[0113] Approximately 60% of persons with cancer will undergo surgery ofsome type, which includes preventative, diagnostic or staging, curativeand palliative surgery. Curative surgery includes resection in which allor part of cancerous tissue is physically removed, excised, and/ordestroyed. Tumor resection refers to physical removal of at least partof a tumor. In addition to tumor resection, treatment by surgeryincludes laser surgery, cryosurgery, electrosurgery, and microscopicallycontrolled surgery (Mohs' surgery). It is further contemplated that thepresent invention may be used in conjunction with removal of superficialcancers, precancers, or incidental amounts of normal tissue. Uponexcision of part of all of cancerous cells, tissue, or tumor, a cavitymay be formed in the body. Treatment may be accomplished by perfusion,direct injection or local application of the area with an additionalanti-cancer therapy, such as with peptides of the invention. Suchtreatment may be repeated, for example, every 1, 2, 3, 4, 5, 6, or 7days, or every 1, 2, 3, 4, and 5 weeks or every 1, 2, 3, 4, 5, 6, 7, 8,9, 10, 11, or 12 months. These treatments may be of varying dosages aswell.

[0114] c) Chemotherapeutic Agents

[0115] Agents that affect DNA function are defined as chemotherapeuticagents, for example, agents that directly cross-link DNA, agents thatintercalate into DNA, and agents that lead to chromosomal and mitoticaberrations by affecting nucleic acid synthesis. Some examples ofchemotherapeutic agents include antibiotic chemotherapeutics, such asDoxorubicin, Daunorubicin, Mitomycin (also known as mutamycin and/ormitomycin-C), Actinomycin D (Dactinomycin), Bleomycin, and Plicomycin;Plant alkaloids such as Taxol, Vincristine, and Vinblastine;miscellaneous agents such as Cisplatin, VP16, Tumor Necrosis Factor;alkylating agents such as, Carmustine, Melphalan (also known as alkeran,L-phenylalanine mustard, phenylalanine mustard, L-PAM, or L-sarcolysin,is a phenylalanine derivative of nitrogen mustard), Cyclophosphamide,Chlorambucil, Busulfan (also known as myleran), Lomustine; and otheragents for example, Cisplatin (CDDP), Carboplatin, Procarbazine,Mechlorethamine, Camptothecin, Ifosfamide, Nitrosurea, Etoposide (VP16),Tamoxifen, Raloxifene, Estrogen Receptor Binding Agents, Gemcitabien,Navelbine, Famesyl-protein transferase inhibitors, Transplatinum,5-Fluorouracil, and Methotrexate, Temazolomide (an aqueous form ofDTIC), or any analog or derivative variant of the foregoing.

[0116] d) Gene Therapy

[0117] In yet another embodiment, the other treatment is a gene therapyin which a therapeutic polynucleotide is administered before, after, orat the same time as a peptide(s) of the invention is administered.Appropriate genes for use according to this embodiment are genesencoding tumor suppressors (p53, Rb, p16), antisense oncogenes (ras,myc, erb), inducers of apoptosis (Bcl-2, Bax) and other such genes.

[0118] e) Other Therapies

[0119] It is contemplated that other agents may be used in combinationwith the present invention to improve the therapeutic efficacy oftreatment. These additional agents include immunomodulatory agents,agents that affect the upregulation of cell surface receptors and GAPjunctions, cytostatic and differentiation agents, inhibitors of celladhesion, agents that increase the sensitivity of the hyperproliferativecells to apoptotic inducers, or other biological agents.Immunomodulatory agents include tumor necrosis factor; interferon alpha,beta, and gamma; IL-2 and other cytokines; F42K and other cytokineanalogs; or MIP-1, MIP-1beta, MCP-1, RANTES, and other chemokines. Inother embodiments, cytostatic or differentiation agents can be used incombination with the present invention to improve theanti-hyperproliferative efficacy of the treatments.

[0120] Another form of therapy for use in conjunction with chemotherapy,radiation therapy or biological therapy includes hyperthermia, which isa procedure in which a patient's tissue is exposed to high temperatures(up to 106° F.). External or internal heating devices may be involved inthe application of local, regional, or whole-body hyperthermia. Localhyperthermia involves the application of heat to a small area, such as atumor. Heat may be generated externally with high-frequency wavestargeting a tumor from a device outside the body. Internal heat mayinvolve a sterile probe, including thin, heated wires or hollow tubesfilled with warm water, implanted microwave antennae, or radiofrequencyelectrodes.

[0121] A patient's organ or a limb is heated for regional therapy, whichis accomplished using devices that produce high energy, such as magnets.Alternatively, some of the patient's blood may be removed and heatedbefore being perfused into an area that will be internally heated.Whole-body heating may also be implemented in cases where cancer hasspread throughout the body. Warm-water blankets, hot wax, inductivecoils, and thermal chambers may be used for this purpose.

[0122] Hormonal therapy may also be used in conjunction with the presentinvention or in combination with any other cancer therapy previouslydescribed. The use of hormones may be employed in the treatment ofcertain cancers such as breast, prostate, ovarian, or cervical cancer tolower the level or block the effects of certain hormones such astestosterone or estrogen. This treatment is often used in combinationwith at least one other cancer therapy as a treatment option or toreduce the risk of metastasis.

[0123] 5. Other Therapeutic Uses

[0124] The peptides of the invention may also be useful as a form ofcontraception, since HA is known to mediate binding of the sperm to theoocyte. Peptides that inhibit HA may inhibit binding between the spermand the oocyte, since such binding requires HA-mediated binding, thuseffectively prevent fertilization, thus effectively preventing formationof the zygote.

[0125] E. Targeting Conjugates Comprising Glycosaminoglyean BindingPeptides

[0126] Targeting other therapeutic agents to sites of inflammation thatare characterized by HA fragments by conjugation to a peptide derivativeof the invention is another ways to use the peptides of the invention inthe therapeutic intervention of inflammation, cancer, or other immunediseases. The agent may be conjugated by a covalent bond or a releasablebond. Several different therapeutic agents are contemplated depending onthe disease the agent is used for. For the treatment of a cancer aradionuclide or a cancer chemotherapeutic agent may be conjugated to apeptide of the invention. The peptide of the invention may be alsoconjugated to a toxin, for example, ricin A chain, cholera toxin,pertussis toxin, etc. Alternatively the present invention anticipatesthe use of an hyaluronidase to reduce the levels of HA.

[0127] In other examples, immunocancer therapy is contemplated, where animmune effector is conjugated or linked to a peptides of the inventionto target and destroy cancer cells. The immune effector may be, forexample, an antibody specific for some marker on the surface of a tumorcell. The antibody alone may serve as an effector of therapy or it mayrecruit other cells to actually effect cell killing. Alternatively, theeffector may be a lymphocyte carrying a surface molecule that interacts,either directly or indirectly, with a tumor cell target. Variouseffector cells include cytotoxic T cells and NK cells.

[0128] In addition, it is contemplated that one may conjugate adiagnostic agent to a peptide of the invention and target this to acancer cell. In context of cancers the diagnostic agent can be agentused to image tumors, for example, through magnetic resonance imaging,x-ray imaging, computerized emission tomography and the like. Elementsparticularly useful in Magnetic Resonance Imaging (“MRI”) include thenuclear magnetic spin-resonance isotopes ¹⁵⁷Gd, ⁵⁵Mn, ¹⁶²Dy, ⁵²Cr, and⁵⁶Fe, with gadolinium often being preferred. Radioactive substances,such as technicium^(99m) or indium¹¹¹, that may be detected using agamma scintillation camera or detector, also may be used. Furtherexamples of metallic ions suitable for use in this invention are ¹²³I,¹³¹I, ¹³¹I, ⁹⁷Ru, ⁶⁷Cu, ⁶⁷Ga, ¹²⁵I, ⁶⁸Ga, ⁷²As, ⁸⁹Zr, and ²⁰¹TI.

[0129] In the context of inflammatory diseases one may conjugate anyother anti-inflammatory agent (see examples in the sections above) toobtain an enhanced anti-inflammatory response.

[0130] F. Pharmaceutical Preparations and Modes of Administration

[0131] Pharmaceutical compositions of the present invention comprise aneffective amount of one or more peptide derivative that binds to andmodulates the function of a glycosaminoglycan or additional agentdissolved or dispersed in a pharmaceutically acceptable carrier. Thephrases “pharmaceutical or pharmacologically acceptable” refers tomolecular entities and compositions that do not produce an adverse,allergic or other untoward reaction when administered to an animal, suchas, for example, a human, as appropriate. The preparation of anpharmaceutical composition that contains at least one peptide derivativeof the invention or additional active ingredient will be known to thoseof skill in the art in light of the present disclosure, as exemplifiedby Remington's Pharmaceutical Sciences, 18th Ed. Mack Printing Company,1990, incorporated herein by reference. Moreover, for animal (e.g.,human) administration, it will be understood that preparations shouldmeet sterility, pyrogenicity, general safety and purity standards asrequired by FDA Office of Biological Standards.

[0132] As used herein, “pharmaceutically acceptable carrier” includesany and all solvents, dispersion media, coatings, surfactants,antioxidants, preservatives (e.g., antibacterial agents, antifungalagents), isotonic agents, absorption delaying agents, salts,preservatives, drugs, drug stabilizers, gels, binders, excipients,disintegration agents, lubricants, sweetening agents, flavoring agents,dyes, such like materials and combinations thereof, as would be known toone of ordinary skill in the art (see, for example, Remington'sPharmaceutical Sciences, 18th Ed., Mack Printing Company, 1990, pp.1289-1329, incorporated herein by reference). Except insofar as anyconventional carrier is incompatible with the active ingredient, its usein the therapeutic or pharmaceutical compositions is contemplated.

[0133] The pharmaceutical formulations of the peptides of the inventionmay comprise different types of carriers depending on whether it is tobe administered in solid, liquid or aerosol form, and whether it need tobe sterile for such routes of administration as injection. The presentinvention can be administered intravenously, intradermally,intraarterially, intraperitoneally, intralesionally, intracranially,intraarticularly, intraprostaticaly, intrapleurally, intratracheally,intranasally, intravitreally, intravaginally, intrarectally, topically,intratumorally, intramuscularly, intraperitoneally, subcutaneously,subconjunctival, intravesicularlly, mucosally, intrapericardially,intraumbilically, intraocularally, orally, topically, locally,inhalation (e.g., aerosol inhalation), injection, infusion, continuousinfusion, localized perfusion bathing target cells directly, via acatheter, via a lavage, in cremes, in lipid compositions (e.g.,liposomes), or by other method or any combination of the forgoing aswould be known to one of ordinary skill in the art (see, for example,Remington's Pharmaceutical Sciences, 18th Ed. Mack Printing Company,1990, incorporated herein by reference).

[0134] The actual dosage amount of a composition of the presentinvention administered to an animal patient can be determined byphysical and physiological factors such as body weight, severity ofcondition, the type of disease being treated, previous or concurrenttherapeutic interventions, idiopathy of the patient and on the route ofadministration. The practitioner responsible for administration will, inany event, determine the concentration of active ingredient(s) in acomposition and appropriate dose(s) for the individual subject.

[0135] In certain embodiments, pharmaceutical compositions may comprise,for example, at least about 0.1% of an active compound. In otherembodiments, the an active compound may comprise between about 2% toabout 75% of the weight of the unit, or between about 25% to about 60%,for example, and any range derivable therein. In other non-limitingexamples, a dose may also comprise from about 1 microgram/kg/bodyweight, about 5 microgram/kg/body weight, about 10 microgram/kg/bodyweight, about 50 microgram/kg/body weight, about 100 microgram/kg/bodyweight, about 200 microgram/kg/body weight, about 350 microgram/kg/bodyweight, about 500 microgram/kg/body weight, about 1 milligram/kg/bodyweight, about 5 milligram/kg/body weight, about 10 milligram/kg/bodyweight, about 50 milligram/kg/body weight, about 100 milligram/kg/bodyweight, about 200 milligram/kg/body weight, about 350 milligram/kg/bodyweight, about 500 milligram/kg/body weight, to about 1000 mg/kg/bodyweight or more per administration, and any range derivable therein. Innon-limiting examples of a derivable range from the numbers listedherein, a range of about 5 mg/kg/body weight to about 100 mg/kg/bodyweight, about 5 microgram/kg/body weight to about 500 milligram/kg/bodyweight, etc., can be administered, based on the numbers described above.

[0136] In any case, the composition may comprise various antioxidants toretard oxidation of one or more component. Additionally, the preventionof the action of microorganisms can be brought about by preservativessuch as various antibacterial and antifungal agents, including but notlimited to parabens (e.g., methylparabens, propylparabens),chlorobutanol, phenol, sorbic acid, thimerosal or combinations thereof.

[0137] The peptides of the invention may be formulated into acomposition in a free base, neutral or salt form. Pharmaceuticallyacceptable salts, include the acid addition salts, e.g., those formedwith the free amino groups of a proteinaceous composition, or which areformed with inorganic acids such as for example, hydrochloric orphosphoric acids, or such organic acids as acetic, oxalic, tartaric ormandelic acid. Salts formed with the free carboxyl groups can also bederived from inorganic bases such as for example, sodium, potassium,ammonium, calcium or ferric hydroxides; or such organic bases asisopropylamine, trimethylamine, histidine or procaine.

[0138] In embodiments where the composition is in a liquid form, acarrier can be a solvent or dispersion medium comprising but not limitedto, water, ethanol, polyol (e.g., glycerol, propylene glycol, liquidpolyethylene glycol, etc.), lipids (e.g. triglycerides, vegetable oils,liposomes) and combinations thereof. The proper fluidity can bemaintained, for example, by the use of a coating, such as lecithin; bythe maintenance of the required particle size by dispersion in carrierssuch as, for example liquid polyol or lipids; by the use of surfactantssuch as, for example hydroxypropylcellulose; or combinations thereofsuch methods. In many cases, it will be preferable to include isotonicagents, such as, for example, sugars, sodium chloride or combinationsthereof.

[0139] In other embodiments, one may use eye drops, nasal solutions orsprays, aerosols or inhalants in the present invention. Suchcompositions are generally designed to be compatible with the targettissue type. In a non-limiting example, nasal solutions are usuallyaqueous solutions designed to be administered to the nasal passages indrops or sprays. Nasal solutions are prepared so that they are similarin many respects to nasal secretions, so that normal ciliary action ismaintained. Thus, in preferred embodiments the aqueous nasal solutionsusually are isotonic or slightly buffered to maintain a pH of about 5.5to about 6.5. In addition, antimicrobial preservatives, similar to thoseused in ophthalmic preparations, drugs, or appropriate drug stabilizers,if required, may be included in the formulation. For example, variouscommercial nasal preparations are known and include drugs such asantibiotics or antihistamines.

[0140] In certain embodiments the peptides of the invention are preparedfor administration by such routes as oral ingestion. In theseembodiments, the solid composition may comprise, for example, solutions,suspensions, emulsions, tablets, pills, capsules (e.g., hard or softshelled gelatin capsules), sustained release formulations, buccalcompositions, troches, elixirs, suspensions, syrups, wafers, orcombinations thereof. Oral compositions may be incorporated directlywith the food of the diet. Preferred carriers for oral administrationcomprise inert diluents, assimilable edible carriers or combinationsthereof. In other aspects of the invention, the oral composition may beprepared as a syrup or elixir. A syrup or elixir, and may comprise, forexample, at least one active agent, a sweetening agent, a preservative,a flavoring agent, a dye, a preservative, or combinations thereof.

[0141] In certain preferred embodiments an oral composition may compriseone or more binders, excipients, disintegration agents, lubricants,flavoring agents, and combinations thereof. In certain embodiments, acomposition may comprise one or more of the following: a binder, suchas, for example, gum tragacanth, acacia, cornstarch, gelatin orcombinations thereof; an excipient, such as, for example, dicalciumphosphate, mannitol, lactose, starch, magnesium stearate, sodiumsaccharine, cellulose, magnesium carbonate or combinations thereof; adisintegrating agent, such as, for example, corn starch, potato starch,alginic acid or combinations thereof; a lubricant, such as, for example,magnesium stearate; a sweetening agent, such as, for example, sucrose,lactose, saccharin or combinations thereof; a flavoring agent, such as,for example peppermint, oil of wintergreen, cherry flavoring, orangeflavoring, etc.; or combinations thereof the foregoing. When the dosageunit form is a capsule, it may contain, in addition to materials of theabove type, carriers such as a liquid carrier. Various other materialsmay be present as coatings or to otherwise modify the physical form ofthe dosage unit. For instance, tablets, pills, or capsules may be coatedwith shellac, sugar or both.

[0142] Additional formulations which are suitable for other modes ofadministration include suppositories. Suppositories are solid dosageforms of various weights and shapes, usually medicated, for insertioninto the rectum, vagina or urethra. After insertion, suppositoriessoften, melt or dissolve in the cavity fluids. In general, forsuppositories, traditional carriers may include, for example,polyalkylene glycols, triglycerides or combinations thereof. In certainembodiments, suppositories may be formed from mixtures containing, forexample, the active ingredient in the range of about 0.5% to about 10%,and preferably about 1% to about 2%.

[0143] Sterile injectable solutions are prepared by incorporating theactive compounds in the required amount in the appropriate solvent withvarious of the other ingredients enumerated above, as required, followedby filtered sterilization. Generally, dispersions are prepared byincorporating the various sterilized active ingredients into a sterilevehicle which contains the basic dispersion medium and/or the otheringredients. In the case of sterile powders for the preparation ofsterile injectable solutions, suspensions or emulsion, the preferredmethods of preparation are vacuum-drying or freeze-drying techniqueswhich yield a powder of the active ingredient plus any additionaldesired ingredient from a previously sterile-filtered liquid mediumthereof. The liquid medium should be suitably buffered if necessary andthe liquid diluent first rendered isotonic prior to injection withsufficient saline or glucose. The preparation of highly concentratedcompositions for direct injection is also contemplated, where the use ofDMSO as solvent is envisioned to result in extremely rapid penetration,delivering high concentrations of the active agents to a small area.

[0144] The composition must be stable under the conditions ofmanufacture and storage, and preserved against the contaminating actionof microorganisms, such as bacteria and fungi. It will be appreciatedthat endotoxin contamination should be kept minimally at a safe level,for example, less that 0.5 ng/mg protein.

[0145] In particular embodiments, prolonged absorption of an injectablecomposition can be brought about by the use in the compositions ofagents delaying absorption, such as, for example, aluminum monostearate,gelatin or combinations thereof

[0146] G. Screening for Compounds that Alter GlycosaminoglycanMetabolism

[0147] The present invention further comprises methods for identifyingmodulators of glycosaminoglycan metabolism. These assays may compriserandom screening of large libraries of candidate substances;alternatively, the assays may be used to focus on particular classes ofcompounds selected with an eye towards structural attributes that arebelieved to make them more likely to modulate glycosaminoglycanmetabolism.

[0148] By metabolism, it is meant that one may assay for changes inglycosaminoglycan synthesis, degradation, transport, or binding. Toidentify a modulator, one generally will determine the metabolism ofglycosaminoglycans in the presence of the candidate substance on a cell,tissue, organ or organism, as compared to metabolism in the absence ofthe candidate substance, a modulator defined as any substance thatalters metabolism. For example, a method generally comprises: providinga candidate modulator; admixing the candidate modulator with a cell,tissue or a suitable experimental animal; measuring one or more aspectsof glycosaminoglycan metabolism in the tissue cell or animal in step;and comparing the aspect measured in step (c) with the aspect of thecell, tissue or animal in the absence of said candidate modulator,wherein a difference between the aspect indicates that said candidatemodulator is, indeed, a modulator of glycosaminoglycan metabolism.

[0149] Assays may be conducted in cell free systems as well.

[0150] It will, of course, be understood that all the screening methodsof the present invention are useful in themselves notwithstanding thefact that effective candidates may not be found. The invention providesmethods for screening for such candidates, not solely methods of findingthem.

[0151] 1. Modulators

[0152] As used herein the term “candidate substance” refers to anymolecule that may potentially modulate glycosaminoglycan metabolism. Thecandidate substance may be a protein or fragment thereof, a smallmolecule, or even a nucleic acid molecule. It may prove to be the casethat the most useful pharmacological compounds will be compounds thatare structurally related to glycosaminoglycans themselves. Using leadcompounds to help develop improved compounds is know as “rational drugdesign” and includes not only comparisons with know inhibitors andactivators, but predictions relating to the structure of targetmolecules.

[0153] The goal of rational drug design is to produce structural analogsof biologically active polypeptides or target compounds. By creatingsuch analogs, it is possible to fashion drugs, which are more active orstable than the natural molecules, which have different susceptibilityto alteration or which may affect the function of various othermolecules. In one approach, one would generate a three-dimensionalstructure for a target molecule, or a fragment thereof. This could beaccomplished by x-ray crystallography, computer modeling or by acombination of both approaches.

[0154] It also is possible to use antibodies to ascertain the structureof a target compound activator or inhibitor. In principle, this approachyields a pharmacore upon which subsequent drug design can be based. Itis possible to bypass protein crystallography altogether by generatinganti-idiotypic antibodies to a functional, pharmacologically activeantibody. As a mirror image of a mirror image, the binding site ofanti-idiotypic would be expected to be an analog of the originalantigen. The anti-idiotype could then be used to identify and isolatepeptides from banks of chemically- or biologically-produced peptides.Selected peptides would then serve as the pharmacore. Anti-idiotypes maybe generated using the methods described herein for producingantibodies, using an antibody as the antigen.

[0155] On the other hand, one may simply acquire, from variouscommercial sources, small molecule libraries that are believed to meetthe basic criteria for useful drugs in an effort to “brute force” theidentification of useful compounds. Screening of such libraries,including combinatorially generated libraries (e.g., peptide libraries),is a rapid and efficient way to screen large number of related (andunrelated) compounds for activity. Combinatorial approaches also lendthemselves to rapid evolution of potential drugs by the creation ofsecond, third and fourth generation compounds modeled of active, butotherwise undesirable compounds.

[0156] Candidate compounds may include fragments or parts ofnaturally-occurring compounds, or may be found as active combinations ofknown compounds, which are otherwise inactive. It is proposed thatcompounds isolated from natural sources, such as animals, bacteria,fungi, plant sources, including leaves and bark, and marine samples maybe assayed as candidates for the presence of potentially usefulpharmaceutical agents. It will be understood that the pharmaceuticalagents to be screened could also be derived or synthesized from chemicalcompositions or man-made compounds. Thus, it is understood that thecandidate substance identified by the present invention may be peptide,polypeptide, polynucleotide, small molecule inhibitors or any othercompounds that may be designed through rational drug design startingfrom known inhibitors or stimulators.

[0157] Other suitable modulators include antisense molecules, ribozymes,and antibodies (including single chain antibodies), each of which wouldbe specific for the target molecule. Such compounds are described ingreater detail elsewhere in this document. For example, an antisensemolecule that bound to a translational or transcriptional start site, orsplice junctions, would be ideal candidate inhibitors.

[0158] In addition to the modulating compounds initially identified, theinventors also contemplate that other sterically similar compounds maybe formulated to mimic the key portions of the structure of themodulators. Such compounds, which may include peptidomimetics of peptidemodulators, may be used in the same manner as the initial modulators.

[0159] An inhibitor according to the present invention may be one whichexerts its inhibitory or activating effect upstream, downstream ordirectly on glycosaminoglycan production. Regardless of the type ofinhibitor or activator identified by the present screening methods, theeffect of the inhibition or activator by such a compound results in analteration in glycosaminoglycan function as compared to that observed inthe absence of the added candidate substance.

[0160] 2. In vitro Cell Free Assays

[0161] A quick, inexpensive and easy assay to run is an in vitro cellfree assay. In the present invention, it only is necessary that a cellfree system include sufficient components that one or more reactionsleading to glycosaminoglycan production be operable.

[0162] 3. In cyto Assays

[0163] The present invention also contemplates the screening ofcompounds for their ability to modulate glycosaminoglycan metabolism incells. Various cell lines can be utilized for such screening assays,including cells specifically engineered for this purpose. Depending onthe assay, culture may be required. The cell is examined using any of anumber of different assays for glycosaminoglycan metabolism.

[0164] 4. In vivo Assays

[0165] In vivo assays involve the use of various animal models. Due totheir size, ease of handling, and information on their physiology andgenetic make-up, mice are a preferred embodiment. However, other animalsare suitable as well, including rats, rabbits, hamsters, guinea pigs,gerbils, woodchucks, cats, dogs, sheep, goats, pigs, cows, horses andmonkeys (including chimps, gibbons and baboons). Assays for modulatorsmay be conducted using an animal model derived from any of thesespecies.

[0166] In such assays, one or more candidate substances are administeredto an animal, and the ability of the candidate substance(s) to alter oneor more characteristics, as compared to a similar animal not treatedwith the candidate substance(s), identifies a modulator. Thecharacteristics may be any of those discussed above with regard to acell (e.g., growth, tumorigenicity, survival), or instead a broaderindication such as behavior, anemia, immune response, etc.).

[0167] Treatment of these animals with test compounds will involve theadministration of the compound, in an appropriate form, to the animal.Administration will be by any route that could be utilized for clinicalor non-clinical purposes, including but not limited to oral, nasal,buccal, or even topical. Alternatively, administration may be byintratracheal instillation, bronchial instillation, intradermal,subcutaneous, intramuscular, intraperitoneal or intravenous injection.Specifically contemplated routes are systemic intravenous injection,regional administration via blood or lymph supply, or directly to anaffected site.

[0168] Determining the effectiveness of a compound in vivo may involve avariety of different criteria. Also, measuring toxicity and doseresponse can be performed in animals in a more meaningful fashion thanin in vitro or in cyto assays.

[0169] H. Examples

[0170] The following examples are included to demonstrate preferredembodiments of the invention. It should be appreciated by those of skillin the art that the techniques disclosed in the examples which followrepresent techniques discovered by the inventor to function well in thepractice of the invention, and thus can be considered to constitutepreferred modes for its practice. However, those of skill in the artshould, in light of the present disclosure, appreciate that many changescan be made in the specific embodiments which are disclosed and stillobtain a like or similar result without departing from the spirit andscope of the invention.

EXAMPLE 1 Chemical Modifications of Pep-1

[0171] Pep-1 was subject to chemical modifications to obtain a morehigh-throughput and cost-effective derivative. The present inventorsdiscovered derivatives that had several desirable properties thatsignificantly enhance their and clinical applicability as well. Forexample, the derivatives were highly water soluble, did not requireadditional solvents for dissolution, higher affinity for theglycosaminoglycan substrate (e.g., HA), and were more effectivebiologically.

[0172] Synthesis of the Pep-1 Dimer. The Pep-1 dimer was synthesized byadding two moles of Pep-1 per mole of succinic acid in 0.1 NHCl, in thepresence of 10-fold molar excess of1-ethyl-3-(3-dimethylaminopropyl)carbodiimide relative to succinic acidand was subsequently purified by size-exclusion column chromatography.

[0173] Properties of the Pep-1 Dimer. The Pep-1 dimer was soluble in theabsence of added solvents such as DMSO and exhibited significantbiological activities to prevent HA-mediated leukocyte adhesion. As thePep-1 dimer formulation has a high solubility it is an ideal choice forlocal administration in therapeutic applications.

[0174] Synthesis of the Pep-1 Tetramer. The Pep-1 tetramer, alsoreferred to as the PEG-conjugate of Pep-1 was prepared by adding 10 to20-fold molar excess amounts of Pep-1 to bis(polyoxyethylenebis[imidazolyl carbonyl]) along with triethylamine. The reaction isallowed to proceed for 5-7 days at 37° C. Afterwards, the reagent isdried under air and resuspended in PBS. Free peptide is removed bydialysis against PBS. Alternatively, the reagent can be directlydialyzed in Spectra/Por® tubing without drying.

[0175] Properties of the Pep-1 Tetramer. This Pep-1 tetramer obtained byPEG-conjugated Pep-1 remained soluble in the absence of added solvents(e.g., DMSO) and exhibited significant biological activities to preventHA-mediated leukocyte adhesion. The PEG-conjugated Pep-1 was also foundto be at least 10-fold more active than Pep-1 in its capacity to inhibitin vitro adhesion of B16F10 melanoma cells to the HA-coated substrates.

[0176] The inventors contemplate that the PEG-conjugated Pep-1 will berelatively resistant to renal filtration and proteolytic digestion ascompared to the original Pep-1. Thus, it is contemplated thatPEG-conjugated Pep-1 will be an ideal formulation for systemicadministration.

EXAMPLE 2 Comparison of Pep-1 and Derivatives

[0177] Pep-1 and its dimeric and tetrameric derivatives were comparedfor their in vivo activities to prevent hapten-triggered LC emigrationfrom the epidermis. Two local injections of the original monomeric Pep-1before DNFB application inhibited LC emigration almost completely whentested at 24 hr after DNFB painting. However, LC began to migrate fromPep-1 -injected skin sites at later time points (48 and 72 hrs afterDNFB application) with the kinetics comparable to those observed in thecontrol group receiving random peptide (RP) injections (FIG. 3). Bymarked contrast, the dimeric form at the same concentration in terms ofthe molarity of the Pep-1 sequence inhibited LC migration significantlyat 24 and 48 hrs after DNFB application. Moreover, the tetrameric formprevented LC migration almost completely at 24 and 48 hr and significant(P<0.01) inhibition was still observed even at 72 hr.

[0178] In other studies that measured inhibition of HA-mediated celladhesion, monomeric Pep-1, at a suboptimal concentration (150 μM),showed only modest inhibition (see FIGS. 2A-B). By contrast, dimericPep-1 and tetrameric Pep-1 at the same concentration in terms of themolarity of the Pep-1 peptide sequence produced significantly (P<0.01)improved inhibition (FIG. 2A), with almost complete inhibition achievedwith the tetrameric form (FIG. 2B). Thus, the multimeric derivatives ofPep-1 demonstrate an enhanced improvement of in vivo pharmacologicalactivities of Pep-1.

EXAMPLE 3 Role in Preventing Metastasis

[0179] Surface expression of particular CD44 isoforms has been observedin many cancers and CD44 inhibitors have been showed to inhibit cancermetastasis in many animal tumor models. Thus, the present inventorsdetermined the potential of Pep-1, an inhibitor of HA function, toprevent tumor metastasis using a fully established model ofexperimentally induced lung metastasis of in vivo infused B16-F10melanoma cells⁷⁶.

[0180] In the model, melanoma cells and Pep-1 (or random peptide controlor PBS alone) were injected together intravenously into syngeneicC57BL/6 mice and lung metastasis was examined macroscopically and byfollowing-up the survival of mice. As reported by other investigators,multiple macroscopic satellite regions were observed in the lung in 10days after tumor inoculation. Simultaneous i.v. injection of Pep-1 (theoriginal monomeric form) together with B16-F10 melanoma cells reducedthe extent of lung metastasis significantly as compared to the RPcontrol panel. Moreover, Pep-1 prolonged the survival of recipientanimals significantly (FIG. 4A). On the other hand, Pep-1 had noapparent effects on the in vitro growth of B16-F10 melanoma cells or onlocal growth in animals following s.c. inoculation (FIG. 4B and FIG.4C). Finally, the B16-F10 melanoma cells showed marked adhesion to theHA-coated plates, and this adhesive interaction was blocked efficientlyby Pep-1, especially in the tetrameric form (FIGS. 2A-B), illustrating amechanism by which Pep-1 interferes with HA-mediated adhesion andtrafficking of melanoma cells. In all four independent experiments,Pep-1 always prevented melanoma metastasis and prolonged the survival ofanimals significantly. The inventors have therefore demonstrated thatlung metastasis of B16-F10 mouse melanoma cell line can be inhibited bysimultaneous administration of Pep-1. These results have revealed anadditional pharmacological activity of Pep-1 to specifically preventtumor metastasis. The inventors will also examine the prophylactic andtherapeutic potentials of multimeric Pep-1 formulations and otherchemically modified Pep-1 derivatives using this animal model ofmelanoma metastasis.

EXAMPLE 4 Impact of Pep-1 on DC-T Cell Interaction

[0181] During antigen-specific interaction DC delivers activationsignals to T cells, leading to differentiation and clonal expansion ofantigen-reactive T cells. At the same time, T cells deliver signals backto DC, leading to “terminal maturation” of DC⁸⁰. To test the potentialimpact of Pep-1 on this bi-directional DC-T cell interaction, thepresent inventors employed a fully characterized in vitro experimentalsystem in which XS52 DC are cultured with a KLH-reactive CD4⁺ Th1 cloneHDK-1 in the presence of antigen (KLH)⁸¹⁻⁸⁵.

[0182] It was found, that Pep-1 inhibited, in a dose-dependent fashion,the secretion of TNFα (by both XS52 and HDK-1 cells), IL-6 (by both celltypes) and interferon-γ (by HDK-1 cells), with almost completeinhibition achieved at 500 μg/ml (FIG. 7B). These results documentanother previously unrecognized function of HA in antigen presentation,namely, high molecular weight HA and/or its degradation products providean essential stimulatory signal during antigen-specific DC-T cellinteraction. Importantly, the present invention also revealed anotherpharmacological activity of Pep-1, which is suppression of antigenpresentation. Pep-1 also inhibited the activation of immunologicallynaive CD4+ T cells by splenic dendritic cells (FIG. 7C).

EXAMPLE 5 Degradation Products of HA

[0183] There has been accumulating evidence in the art, to support thenew concept that degradation products of HA (i.e., HA fragments), justlike fragments of other extracellular matrix components (e.g., collagensand fibronectin), act as pro-inflammatory mediators. The presentinventors have demonstrated that HA fragments trigger cytokineproduction from DC, e.g., IL-6 and TNF alpha production by the XS52 DCline. In addition, the inventors have demonstrated that theglycosaminoglycan peptide-inhibitors of the invention, including Pep-1and its derivatives, significantly block the production of bothcytokines by XS52 cells following stimulation with HA fragments.

[0184] To test the impact of Pep-1 on HA degradation, the presentinventors developed a 96 well-based assay in which biotinylated HA wasimmobilized onto the plates and treated with HAase. The amounts of HAthat remained attached to the wells was then determined by addition ofstreptavidin-conjugated alkaline phosphatase (FIG. 5A). Pep-1 failed toaffect the degradation of HA substrates in this assay, while Pep-1 atthe same concentration blocked BW5147 cell adhesion to the same HAsubstrate prepared in parallel (FIG. 5B). This indicates that Pep-1 isnot merely acting as a non-specific masking reagent for HA.

[0185] To test the potential impact of Pep-1 on pro-inflammatoryactivities of HA fragments, low molecular weight HA fragments wereprepared by sonication and enzymatic digestion of HA and the resultingfragments were added to the immature DC line XS52. The inventors HAfragment preparation triggered the secretion of IL-6 and TNFα by XS52cells (FIG. 5C). Importantly, Pep-1, but not RP, inhibited the secretionof both cytokines almost completely. These results have unveiled yetanother pharmacological activity of Pep-1, that comprises neutralizingthe DC-stimulatory potential of HA fragments.

[0186] A critical question concerns the physiological significance ofthe above finding. Topical application of haptens is known to triggernot only LC migration from the epidermis, but also their maturation.Thus, the impact of locally injected Pep-1 on DNFB-induced LC maturationwas tested in situ by testing surface expression of CD86 by IA⁺epidermal cells (i.e., LC). LC isolated from vehicle-treated skinexpressed CD86 at minimal levels, whereas LC from DNFB-treated skinshowed significantly elevated CD86 expression (FIG. 6A). Importantly,two local injections of Pep-1, but not RP, prevented this DNFB-inducedLC maturation in three independent experiments (FIGS. 6B and 6C). Notonly do these results support the inventors hypothesis that Pep-1 can beused to prevent DC maturation in vivo, they also imply that HAdegradation must take place in the epidermal compartment in response tohapten application.

EXAMPLE 6 HA Expression and Metabolism

[0187] The inventors observations, described in Example above, indicatethe potential of keratinocytes and DC to synthesize and degrade HAeither spontaneously or upon activation. Three HA synthases (I, II, andIII) and two HAases (I and III) have been cloned in mice, and thepresent inventors have identified a mouse homologue of human HAase IIfrom the EST database. In RT-PCR experiments, the present inventorsdetected constitutive mRNA expression of HA synthases-2 and-3 andHAases-1, 2, and 3 in mouse skin (see Table 2). PCR signals for the sameenzymes were detected in the Pam 212 keratinocyte, XS52 DC, XS106 DC,and NS46 fibroblast lines.

[0188] The data in Table 2 depict the RT-PCR results, where total RNAwas isolated from BALB/c mouse skin or the indicated cell lines,reverse-transcribed and examined for expression of the indicated mRNA byPCR using the following primer pairs: 5′-TATCCAACCGGCCATTCAATCACTG-3′(SEQ ID NO:3) and 5′-ATACCCCGCTTGTCACACCACTTG-3′ (SEQ ID NO:4) forHAase-1; 5′-CATGTTCACTGGCCGACCCTTTGT-3′ (SEQ ID NO:5) and5′-TCGCCACCCCAGCCCAGATAGC-3′ (SEQ ID NO:6) for HAase-2; and5′-CCTAGGCCTAATGATGGTG-3′ (SEQ ID NO:7) and 5′-GCTAGTATGGGCTTTGTGG-3′(SEQ ID NO:8) for HAase-3; 5′-CTACGGGCGCTGTCGGTGAAGGT-3′ (SEQ ID NO:9)and 5′-CGGGGACATAGTTAGCAGCCAGTT-3′ (SEQ ID NO:10) for HA synthase-1;5′-TGGAACACCGGAAAATGAAGAAG-3′ (SEQ ID NO:11) and5′-GACCGAGCCGTGTATTTAGTTGC-3′ (SEQ ID NO:12) for HA synthase 2; and5′-CCATGAGGCGGGTGAAGGAGAG-3′ (SEQ ID NO:13) and5′-ATGCGGCCACGGTAGAAAAGTTGT-3′ (SEQ ID NO:14) for HA synthase-3′;

[0189] No PCR signals were detected for HA synthase-1. The data shown inTable 2 are PCR products after 35 cycles of amplification visualizedwith ethidium bromide.

[0190] Detection of mRNA for hyaluronidases and for hyaluronan synthasesin keratinocytes demonstrate that several enzymes involved in HAmetabolism are expressed at least at the mRNA levels by the Pam 212keratinocyte line and in mouse skin. This indicates that keratinocytes(and other cell types in the skin) can potentially produce HA fragmentsin response to environmental stimuli. These observations also validatethe topical application of Pep-1 to neutralize the pro-inflammatoryactivities of locally produced HA fragments. TABLE 2 mRNA Expression ofHA Synthases and HAases by Diverse Cell Types in Skin No Skin Pam212 KCXS52 DC XS106 DC NS47 FB Template HAse 1 + ++ ++ ++ ++ − HAse 2 + ++ ++++ ++ − HAse 3 ++ ++ ++ ++ ++ − HA Synthase 2 + ++ + ++ + − HA Synthase3 + + ++ ++ + − β-Actin ++ ++ ++ ++ ++ −

[0191] Metabolic labeling of XS106 DC with ³H-glucosamine revealed thathyaluronidase is incorporated most predominantly into membrane (FIG.8B). Moreover, Pep-1 in a biotinylated form bound to the surfaces ofXS106 DC (FIG. 8C). A 12-mer control peptide (SATPASAPYPLA), termed“random peptide” (RP), showed no significant binding, and Pep-1 bindingwas abolished almost completely by hyaluronidase pretreatment of DC(FIG. 8C). Pep-1 also bound to all other tested DC preparations,including XS52 DC, splenic DC, and bone marrow-derived DC (FIG. 8D). Indouble-staining experiments, in which XS106 DC were double-stained withbiotinylated Pep-1 or RP followed by FITC-conjugated streptavidin andwith PE-conjugated anti-CD44 mAb, biotinylated Pep-1 showed diffuse anduniform binding to the cell bodies, whereas phycoerythrin(PE)-conjugated anti-CD44 mAb exhibited somewhat distinct bindingprofiles.

EXAMPLE 7 Function of HA in T Cell Communication and Proliferation

[0192] CD4⁺ T cells purified from DO11.10 mice (BALB/c background)express the transgenic T cell receptor α- and β-chains specific forovalbumin (OVA) peptide 323-339, providing an unique opportunity tostudy the potential contribution of HA being expressed on DC to theirintrinsic capacity to activate naive T cells. DO11.10 T cells showedrobust ³H-thymidine uptake when stimulated by splenic DC (isolated fromBALB/c mice) in the presence of OVA₃₂₃₋₃₃₉ (FIG. 9A). By contrast, nosignificant proliferation was observed in the absence of DC or antigen,indicating DC-dependency and antigen-specificity.

[0193] Bovine proteoglycan (bPG), which is known to bind to and inhibitthe function of HA specifically, abolished the proliferative responsesof DO11.10 T cells completely, whereas it showed no inhibitory potentialafter heat inactivation (FIG. 9A). DC-dependent, antigen-specific T cellproliferation was also inhibited by Pep-1 (FIG. 9B). Pep-1 moleculeswere conjugated to a tetravalent polyethylene glycol (PEG) derivative byincubating a 40-fold molar excess of Pep-1 or RP with bis(polyethylenebis[imidazolyl carbonyl]) in DMSO containing 0.5% triethylamine for 7days. Reactions were terminated by extensive dialysis against dH₂O orPBS and the density of substitution was estimated by the BCA assay (todetermine peptide content) and dry weight analysis. The resultingpreparations, which showed an estimated molecular ratio of Pep-1/PEG of2.3-2.9 (data not shown), were significantly more potent than theoriginal Pep-1 preparations in blocking both cell adhesion to HA-coatedplates (FIG. 2C) and cellular binding of soluble HA (FIG. 9D). ThePEG-conjugated Pep-1 formulation inhibited splenic DC-dependent DO11.10T cell proliferation almost completely (80-100%) even at lowerconcentrations (50-150 μM in terms of the molarity of the 12-mer Pep-1sequence) (FIG. 9E). PEG-Pep-1, but not PEG-RP, also efficientlyinhibited DO11.10 T cell proliferation triggered by bone marrow-derivedDC (FIG. 9F). Anti-CD44 mAb (KM81) affected DC-induced T cellproliferation only partially even at the concentration 10 times higherthan that required to completely block T cell adhesion to HA-coatedplates (FIG. 9G). This may suggest that CD44 is not the only relevantreceptor for DC-associated HA. Alternatively, T cells may employdifferent CD44 isoforms to recognize HA moieties expressed on DCsurfaces versus HA molecules immobilized on culture plates.Nevertheless, the data indicate that blockade of DC-associated HA witheach tested inhibitor markedly diminishes the intrinsic ability of DC totrigger T cell proliferation.

[0194] To test whether PEG-Pep 1 inhibited T cell proliferation bycausing cell death, the Inventors examined T cell proliferation by usinga fluorescence dye 5,6-carboxy-succinimidyl-fluorescein-ester (CSFE).When stimulated by DC in the presence of OVA₃₂₃₋₃₃₉, CSFE-labeledDO11.10 T cells showed marked reduction in the fluorescence intensities,reflecting their progressive mitosis (FIG. 10A). By contrast, nosignificant mitotic activity was observed in the absence of antigen.Consistent with our observations in ³H-thymidine uptake assays, T cellmitosis was prevented completely by PEG-Pep-1, but not PEG-RP. It shouldbe noted that the Inventors examined CSFE profiles within the propidiumiodide (PI)-negative populations. Thus, PEG-Pep-1 inhibits the growth ofT cells without affecting their viability. PEG-Pep-1 showed nosignificant effect on the viability of DC, as measured by PI uptakewithin the CD11c⁺ populations. To test the possibility that PEG-Pep 1may inpair the overall ability of DC to deliver T cell activationsignals, the Inventors measured cytokine release from T cells. DO11.10 Tcells secreted significant amounts of IL-2 and interferon-γ (IFNγ) onlywhen stimulated by DC in the presence of antigen (FIG. 10B). Productionof both cytokines was inhibited efficiently by PEG-Pep-1, but not byPEG-RP, suggesting that antigen-specific DC-T cell communication isbroadly impaired. To test whether PEG Pep 1 might physically block thecontact between DC and T cells during antigen presentation, theInventors examined DC-T cell cluster formation. DO11.10 T cells werecultured with bone marrow-derived DC and/or OVA₃₂₃₋₃₃₉ in the presenceof PEG-Pep-1 (60 μM), PEG-RP, or PBS alone. DO11.10 T cells formedrelatively large clusters with DC after 16 hr co-culturing in thepresence of OVA₃₂₃₋₃₃₉ By contrast, only a few clusters were observed inthe absence of antigen. This antigen-specific DC-T cell clusterformation was blocked almost completely by PEG-Pep-1, but not by PEG-RP.Thus, HA molecules expressed on DC facilitate the establishment ofphysical contact and the subsequent intercellular communication betweenDC and T cells during antigen presentation.

[0195] The Inventors developed an in vivo assay for testinghoming-independent DC-T cell interaction. This method involvesintraperitoneally injecting OVA₃₂₃₋₃₃₉-pulsed DC and CSFE-labeledDO11.10 T cells and examining the CSFE profiles of the cells recoveredfrom the peritoneal cavity. Significant reduction in the fluorescenceintensities was detected in those animals receiving antigen-pulsed DC,but not PBS-pulsed DC, validating antigen-specificity of the new assaysystem. Intraperitoneal administration of PEG-Pep-1, but not PEG-RP,appeared to inhibit the mitosis of DO11.10 T cells (FIG. 11A). In fact,the PEG-Pep-1 treatment panel showed a statistically significantreduction in the % of dividing cells in two independent experiments, ascompared to the non-treatment panel and to the second control panelreceiving PEG-RP injection (FIG. 11B).

[0196] The Inventors have identified a capability of DC toconstitutively express mRNAs for various enzymes involved in HAsynthesis and degradation, actively synthesize HA, preferentiallyincorporate newly synthesized HA into membrane fractions, and uniformlyexpress HA on their surfaces. With respect to the function ofDC-associated HA, DC-dependent, antigen-specific proliferation of CD4⁺ Tcells was inhibited by two distinct HA inhibitors (bPG and Pep-1).Moreover, the oligomeric Pep-1 formulation blocked almost completelyantigen-specific DC-T cell cluster formation, DC-induced cytokineproduction by T cells, and in vivo DC-T cell interaction. Thus, it isproposed proposed that HA molecules expressed on DC mediateintercellular communication between DC and T cells during antigenpresentation.

EXAMPLE 8 Method of Screening for an Inhibitor ofGlycosaminoglycan-mediated Reactions

[0197] Agents, for example other peptides, macromolecules or smallmolecules, that bind glycosaminoglycans, in particular HA, can bediscovered using various screening methods. For example beads or othersupports treated linked to HA are pre-incubated with agents of interest.Peptides comprising the general formula (Z)_(n)X(Y)_(m), SEQ ID NO: 1 orSEQ ID NO: 2, multimer peptides comprising the general formula(Z)_(n)X(Y)_(m), or peptides of the sequence of SEQ ID. NO: 15 whichbind HA are labeled with ¹²⁵I and tested at 50 μg/ml for the binding tothe pre-incubated HA-coated beads. The peptides are radiolabeld at theN-terminus using the Bolton-Hunter reagent and incubated for 2 hours at4° C. Following incubation, the beads are washed three times and thelevels of binding detected, and compared to levels of binding detectedin control experiments. Agents that bind HA can be identified becausethey inhibit the binding of the radiolabeled peptide, and this resultsin a lower level of binding detected in the experimental compared to thecontrol experiments. Similar assays may be peformed using an unlabeledpeptides, wherein the binding of peptides to pre-incubated andexperimental samples is detected and measured using a specific antibodyagainst the peptide.

[0198] Agents can be further assessed for their ability to inhibitHA-mediated reactions using protocols described in the disclosure.

[0199] All of the compositions and/or methods disclosed and claimedherein can be made and executed without undue experimentation in lightof the present disclosure. While the compositions and methods of thisinvention have been described in terms of preferred embodiments, it willbe apparent to those of skill in the art that variations may be appliedto the compositions and/or methods and in the steps or in the sequenceof steps of the method described herein without departing from theconcept, spirit and scope of the invention. More specifically, it willbe apparent that certain agents which are both chemically andphysiologically related may be substituted for the agents describedherein while the same or similar results would be achieved. All suchsimilar substitutes and modifications apparent to those skilled in theart are deemed to be within the spirit, scope and concept of theinvention as defined by the appended claims.

1 17 1 12 PRT Artificial Sequence Synthetic polypeptide, wherein Xaa isany amino acid. 1 Gly Ala Xaa Trp Gln Phe Xaa Ala Leu Thr Val Xaa 1 5 102 12 PRT Mus musculus 2 Gly Ala His Trp Gln Phe Asn Ala Leu Thr Val Arg1 5 10 3 25 DNA Mus musculus 3 tatccaaccg gccattcaat cactg 25 4 24 DNAMus musculus 4 ataccccgct tgtcacacca cttg 24 5 24 DNA Mus musculus 5catcttcact ggccgaccct ttgt 24 6 22 DNA Mus musculus 6 tcgccaccccagcccagata gc 22 7 19 DNA Mus musculus 7 cctaggccta atgatggtg 19 8 19DNA Mus musculus 8 gctagtatgg gctttgtgg 19 9 23 DNA Mus musculus 9ctacgggcgc tgtcggtgaa ggt 23 10 24 DNA Mus musculus 10 cggggacatagttagcagcc agtt 24 11 23 DNA Mus musculus 11 tggaacaccg gaaaatgaag aag23 12 23 DNA Mus musculus 12 gaccgagccg tgtatttagt tgc 23 13 22 DNA Musmusculus 13 ccatgaggcg ggtgaaggag ag 22 14 24 DNA Mus musculus 14atgcggccac ggtagaaaag ttgt 24 15 16 PRT Artificial Sequence Syntheticpolypeptide 15 Gly Ala His Trp Gln Phe Asn Leu Ala Thr Val Arg Gly GlyGly Ser 1 5 10 15 16 15 PRT Artificial Sequence Synthetic polypeptide 16Arg Arg Gly Ala His Trp Gln Phe Asn Ala Leu Thr Val Arg Arg 1 5 10 15 1713 PRT Artificial Sequence Synthetic polypeptide 17 Arg Arg His Trp GlnPhe Asn Ala Leu Thr Val Arg Arg 1 5 10

What is claimed is:
 1. An artificial peptide multimer, comprising astructure: (Z)_(n)X(Y)_(m) wherein X is any naturally occurring aminoacid and Z and Y are each independently selected from the groupconsisting of an aliphatic amino acid and a polar aliphatic amino acid,and n and m are each independently an integer in the range of from 6 to30; wherein the peptide multimer has a binding affinity K_(a) of 5×10⁵l/mol or more relative to a naturally occurring glycosaminoglycan insitu.
 2. The artificial peptide multimer of claim 1, wherein saidmultimer comprises two or more peptide units having the same amino acidsequence and the K_(a) is 1×10⁶ l/mol or more.
 3. The artificial peptidemultimer of claim 1, wherein said multimer comprises two or more peptideunits having different amino acid sequences and the K_(a) is 1 x 10⁶l/mol or more.
 4. The artificial peptide multimer of claim 1, whereinsaid multimer is a dimer.
 5. The artificial peptide multimer of claim 1,wherein said multimer is a tetramer.
 6. The artificial peptide multimerof claim 1, wherein said glycosaminoglycan is selected from the groupconsisting of hyaluronic acid, a salt of hyaluronic acid, chondroitinsulfate, chondroitin sulfate C, dermatan sulfate, heparin, keratansulfate, keratosulfate, chitin, chitosan 1 and chitosan 2, and the K_(a)is 1×10⁸ l/mol or more.
 7. The artificial peptide multimer of claim 1,wherein said structure is connected to a second structure by one or morelinker molecules.
 8. The artificial peptide multimer of claim 7, whereinsaid one or more linker molecules comprise one or more amino acids. 9.The artificial peptide multimer of claim 7, wherein said linker is anon-peptide linker.
 10. The artificial peptide multimer of claim 9,wherein said non-peptide linker comprises succinic acid.
 11. Theartificial peptide multimer of claim 9, wherein said non-peptide linkercomprises polyethylene glycol (PEG).
 12. The artificial peptide multimerof claim 1, wherein Z and Y are the same and n and m are each
 6. 13. Theartificial peptide multimer of claim 1, wherein said structure furthercomprises an N- or C-terminal extension W_(p), wherein W is any basic orneutral amino acid, and p is an integer between 3 and
 13. 14. Theartificial peptide multimer of claim 13, wherein W is selected from thegroup consisting of glycine and arginine and wherein the peptide subunitcomprises a terminal serine.
 15. The artificial peptide multimer ofclaim 1, wherein said structure is chemically modified, wherein thechemical modification is selected from the group consisting of amidationand treatment with polyethylene glycol, and wherein one or more of saidamino acids is a D-isomer.
 16. The peptide multimer of claim 1, whereineach structure comprises the peptide sequence GAXWQFXALTVXGGGS, whereinX is any amino acid.
 17. A pharmaceutical composition comprising: (a) anartificial peptide multimer of claim 1; and (b) a pharmaceuticallyacceptable carrier.
 18. A method of inhibiting aglycosaminoglycan-mediated reaction comprising administering to asubject an agent having the ability to bind a glycosaminoglycan orfragment thereof with a binding affinity of K_(a) of 5×10⁵ l/mol or morerelative to a naturally occurring glycosaminoglycan in situ, whereinadministering the agent inhibits the glycosaminoglycan-mediatedreaction.
 19. The method of claim 18, wherein theglycosaminoglycan-mediated reaction is an inflammatory reaction.
 20. Themethod of claim 18, wherein said agent inhibits an interaction of anantigen presenting cell and a T cell.
 21. The method of claim 20,wherein said antigen presenting cell is a dendritic cell.
 22. The methodof claim 18, wherein administration is selected from the groupconsisting of topical, local, regional and systemic administration. 23.The method of claim 18, wherein the agent is an an peptidic compoundcomprising a structure: (Z)_(n)X(Y)_(m) wherein X is any naturallyoccurring amino acid and Z and Y are each independently selected fromthe group consisting of an aliphatic amino acid and a polar aliphaticamino acid, and n and m are each independently an integer in the rangeof from 6 to 30; wherein the artificial peptide has a binding affinityK_(a) of 5×10⁵ l/mol or more relative to a naturally occurringglycosaminoglycan in situ.
 24. The method of claim 18, wherein theglycosaminoglycan-mediated reaction is tumorigenesis.
 25. The method ofclaim 24, wherein the glycosaminoglycan-mediated reaction is cancermetastasis.
 26. The method of claim 25, wherein said tumorigensis istumorigenesis of brain cancer, lung cancer, throat cancer, esophagealcancer, cancer of the head and neck, skin cancer, breast cancer, stomachcancer, colon cancer, cancer of the rectum, cervical cancer, prostatecancer, ovarian cancer, liver cancer, pancreatic cancer or a cancer ofthe blood.
 27. The method of claim 23, wherein said peptide isadministered in conjunction with a second anti-cancer therapy.
 28. Amethod of treating or preventing cancer comprising administering to asubject an agent that modulates the synthesis, secretion or degradationof a glycosaminoglycan.
 29. The method of claim 28, wherein the agent isselected from the group consisting of hyaluronidase, β-D-glucuronidaseand β-N-acetyl-D-hexosaminidase.
 30. The method of claim 18, whereinsaid glycosaminoglycan is hyaluronic acid, chondroitin sulfate,chondroitin sulfate C, dermatan sulfate, heparin, keratan sulfate,keratosulfate, chitin, chitosan 1 and chitosan
 2. 31. A method ofscreening for an inhibitor of glycosaminoglycan mediated reactions,comprising: (a) contacting said cell that expresses a glycosaminoglycanwith a candidate substance; and (b) measuring the synthesis, secretion,degradation, surface expression or function of said glycosaminoglycan,wherein a change in the synthesis, secretion, degradation, surfaceexpression or function of a glycosaminoglycan, as compared to a similarcell not treated with said candidate substance, identifies saidcandidate substance as an an inhibitor of glycosaminoglycan mediatedreaction.